Nucleotide sequences and corresponding polypeptides conferring modulated growth rate and biomass in plants grown in saline and oxidative conditions

ABSTRACT

The present invention relates to isolated nucleic acid molecules and their corresponding encoded polypeptides able confer the trait of improved plant size, vegetative growth, growth rate, seedling vigor and/or biomass in plants challenged with saline and/or oxidative stress conditions. The present invention further relates to the use of these nucleic acid molecules and polypeptides in making transgenic plants, plant cells, plant materials or seeds of a plant having plant size, vegetative growth, growth rate, seedling vigor and/or biomass that are improved in saline and/or oxidative stress conditions with respect to wild-type plants grown under similar conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of co-pending application Ser. No.13/465,841, filed on May 7, 2012, which is a Divisional of applicationSer. No. 11/858,117, filed on Sep. 19, 2007 (abandoned), which is aContinuation in Part of Application No. PCT/US2007/06544, filed on Mar.14, 2007, and claims priority under 35 U.S.C § 120 of Application No.PCT/US2007/06544, which claims priority under 35 U.S.C. § 119 of U.S.Provisional No. 60/782,735, filed on Mar. 14, 2006, the contents of eachof which are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to isolated nucleic acid molecules andtheir corresponding encoded polypeptides able to enhance plant growthunder saline and/or oxidative stress conditions. The present inventionfurther relates to using the nucleic acid molecules and polypeptides tomake transgenic plants, plant cells, plant materials or seeds of a planthaving improved growth rate, vegetative growth, seedling vigor and/orbiomass under saline and/or oxidative stress conditions as compared towild-type plants grown under similar conditions.

BACKGROUND

Plants specifically improved for agriculture, horticulture, biomassconversion, and other industries (e.g. paper industry, plants asproduction factories for proteins or other compounds) can be obtainedusing molecular technologies. As an example, great agronomic value canresult from enhancing plant growth in saline and/or oxidative stressconditions.

Salinity

A wide variety agriculturally important plant species demonstratesignificant sensitivity to saline and/or oxidative stress conditions.Upon salt concentration exceeding a relatively low threshold, manyplants suffer from stunted growth, necrosis, and death that results inan overall stunted appearance and reduced yields of plant material,seeds, fruit and other valuable products. Physiologically, plantschallenged with salinity experience disruption in ion and waterhomeostasis, inhibition of metabolism, and damage to cellular membranesthat result in developmental arrest and cell death (Huh et al. (2002)Plant J, 29(5):649-59).

In many of the world's most productive agricultural regions,agricultural activities themselves lead to increased water and soilsalinity, which threatens their sustained productivity. One example iscrop irrigation in arid regions that have abundant sunlight. Afterirrigation water is applied to cropland, it is removed by the processesof evaporation and transpiration. While these processes remove waterfrom the soil, they leave behind dissolved salts carried in irrigationwater. Consequently, soil and groundwater salt concentrations build overtime, rendering the land and shallow groundwater saline and thusdamaging to crops.

In addition to human activities, natural geological processes havecreated vast tracts of saline land that would be highly productive ifnot saline. In total, approximately 20% of the irrigated lands arenegatively affected by salinity. (Yamaguchi and Blumwald, 2005, Trendsin Plant Science, 10: 615-620). For these and other reasons, it is ofgreat interest and importance to identify genes that confer improvedsalt tolerance characteristics to thereby enable one to createtransgenic plants (such as crop plants) with enhanced growth and/orproductivity characteristics in saline conditions.

Despite this progress, today there continues to be a great need forgenerally applicable processes that improve forest or agricultural plantgrowth to suit particular needs depending on specific environmentalconditions. To this end, the present invention is directed toadvantageously manipulating plant tolerance to salinity in order tomaximize the benefits of various crops depending on the benefit sought,and is characterized by expression of recombinant DNA molecules inplants. These molecules may be from the plant itself, and simplyexpressed at a higher or lower level, or the molecules may be fromdifferent plant species.

Oxidative Stress

Plants lead a sessile lifestyle and so are generally destined to residewhere their seed germinates. Consequently, they can be exposed tounfavorable environmental conditions arising from weather, pollution andlocation. Stress conditions, such as extremes in temperature, droughtand desiccation, salinity, soil nutrient content, heavy metals, UVradiation, pollutants such as ozone and SO₂, mechanical stress, highlight and pathogen attack, have a large impact on plant growth anddevelopment. These types of stress exposure induce formation of toxicoxygen species, which are generated in all aerobic cells and areassociated with oxidative damage at the cellular level. Several recentlypublished reports have characterized toxic oxygen species generation andthe subsequent oxidative damage caused by abiotic stresses (seeLarkindale and Knight (2002); Borsani et al. (2001); Lee et al (2004);Aroca et al (2005); Luna et al (2005); and Noctor et al (2002)).

The toxic oxygen species are referred to as reactive oxygen species(ROS), reactive oxygen intermediates (ROI) or activated oxygen species(AOS) and are partially reduced or activated derivatives of oxygen.ROS/ROI/AOS include the oxygen-centered superoxide (O₂) and hydroxyl(.OH) free radicals as well as hydrogen peroxide (H₂O₂), nitric oxide(NO) and O₂ ¹. These oxygen species are generated as byproducts fromreactions that occur during photosynthesis, respiration andphotorespiration, and are predominantly formed in the chloroplasts,mitochondria, endoplasmic reticulum, microbodies (e.g. peroxisomes andglyoxysomes), plasma membranes and cell walls. While the toxicity of O₂⁻ and H₂O₂ themselves is relatively low, their metal-dependentconversion to highly toxic .OH is thought to be responsible for themajority of the biological damage associated with these molecules.

Oxidative stress damages cell structure and affects cell metabolism andcatabolism. Membrane lipids are subject to oxidation by ROS/ROI/AOS,resulting in accumulation of high molecular weight, cross-linked fattyacids and phospholipids. Oxidative attack on proteins results insite-specific amino acid modifications, fragmentation of the peptidechain, aggregation of cross-linked reaction products, altered electricalcharge and increased susceptibility to proteolysis, all of whichfrequently leads to elimination of enzyme activity. ROS/ROI/AOS thatgenerate oxygen free radicals, such as ionizing radiation, also inducenumerous lesions in DNA at both the sugar and base moieties which causedeletions, mutation and other lethal genetic effects such as basedegradation, single strand breakage and cross-linking to proteins.Morphologically, the adverse effects of high levels of ROS accumulationare manifested as stunted growth and necrotic lesions.

Although capable of producing damage, ROS/ROI/AOS are also keyregulators of metabolic and defense pathways, playing roles as signalingor secondary messenger molecules. For example, pathogen-inducedROS/ROI/AOS production is critical in disease resistance where thesemolecules are involved at three different levels: penetrationresistance, hypersensitive response (HR) and systemic acquiredresistance (Levine et al. (1994); Lamb and Dixon (1997); Zhou et al.(2000); Aviv et al. (2002)). In penetration resistance, ROS/ROI/AOSfunction by reinforcing cell walls through polyphenolic cross-linking.With respect to hypersensitive response, H₂O₂ is an active signalingmolecule whose effect is dose dependent. At high dosages, H₂O₂ triggershypersensitive cell death and thus restricts the pathogen to localinfection sites (Lamb and Dixon (1997)) while low dosages block cellcycle progression (Reichheld et al. (1999)) and signal secondary walldifferentiation (Potikha et al. (1999)). Lastly, ROS/ROI/AOS moleculesplay a role in broad-spectrum systemic acquired disease resistance bytriggering micro-HR systematically after the first pathogen inoculation.

In the signal cascades leading to oxidative stress, salicylic acid (SA)has been identified as an important signaling molecule to mediateROS/ROI/AOS accumulation in various stress conditions, such as salt andosmotic stress (Borsani et al. (2001)), drought (Senaratna et al.(2000)), heat (Dat et al. (1998)), cold (Scott et al. (2004)), UV-light(Surplus et al. (1998)), paraquat (Kim et al. (2003)) and diseaseresistance against different pathogens (Zhou et al. (2004)). High levelsof SA induce H₂O₂ production as well as cell death.

Several signaling components required for SA-mediated ROS/ROI/AOSaccumulation and gene expression have been characterized. For example,NPR1 is required for SA-induced PR gene expression and diseaseresistance (Cao et al. (1994)). The mutations in eds1 and eds5 blockSA-mediated signaling and enhance disease susceptibility (Rusterucci etal. (2001)). Over-expression of NahG in various plant species alsosuppresses SA-induced responses to both abiotic and biotic stresses(Delaney et al. (1994)). Recently, Scott and colleagues (2004) reportedthat chilling treatment induced accumulation of SA in Arabidopsis andthe degradation of SA by overexpression of NahG enhanced cold tolerancein a transgenic plant.

SA, as a phytohormone, also promotes early flowering (Martinez et al.(2004)). SA at various levels may play different roles in plant growthand stress responses. However, most of the time, the increased toleranceto high levels of SA appears to be beneficial, since it reduces the sideeffects of SA accumulation while stimulating SA-mediated stressresponses.

Similarly, NO is capable of generating ROS/ROI/AOS and is a plantsignaling molecule involved in the regulation of seed germination,stomatal closure (Mata and Lamattina (2001); Desikan et al (2002)),flowering time (He et al. (2004)), antioxidant reactions to suppresscell death (Beligni et al. (2002)) and tolerance to biotic and abioticstress conditions (Mata and Lamattina (2001)). While the effects of NOcan be mimicked through the application of sodium nitroprusside (SNP),endogenous NO production in plants results from the activity of a nitricoxide synthase that uses L-arginine (Guo et al. (2003)) as well asnitrate reductase-mediated reactions (Desikan et al (2002)). NO canreact with redox centers in proteins and membranes, thereby causing celldamage and inducing cell death.

In order to control the two-fold nature of ROS/ROI/AOS molecules, plantshave developed a sophisticated regulatory system which involves bothproduction and scavenging of ROS/ROI/AOS in cells. During normal growthand development, this pathway monitors the level of ROS/ROI/AOS producedby metabolism and controls the expression and activity of ROS/ROI/AOSscavenging pathways. The major ROS/ROI/AOS scavenging mechanisms includethe action of the superoxide dismutase (SOD), ascorbate perioxidase(APX) and catalase (CAT) enzymes as well as nonenzymatic components suchas ascorbic acid, α-tocopherol and glutathione.

The antioxidant enzymes are believed to be critical components inpreventing oxidative stress, in part because pretreatment of plants withone form of stress, and which induces expression of these enzymes, canincrease tolerance for a different stress (cross-tolerance) Allen(1995)). In addition, plant lines selected for resistance to herbicidesthat function by inducing ROS/ROI/AOS generally have increased levels ofone or more of these antioxidant enzymes and also exhibitcross-tolerance (Gressel and Galun (1994)).

Plant development and yield depend on the ability of the plant to manageoxidative stress, whether it is via the signaling or the scavengingpathways. Consequently, improvements in a plant's ability to withstandoxidative stress, or to obtain a higher degree of cross-tolerance onceoxidative stress has been experienced, has significant value inagriculture. The sequences and methods of the invention provide themeans by which tolerance to oxidative stress can be improved, either viathe signaling or the scavenging pathways.

The availability and sustainability of a stream of food and feed forpeople and domesticated animals has been a high priority throughout thehistory of human civilization and lies at the origin of agriculture.Specialists and researchers in the fields of agronomy science,agriculture, crop science, horticulture, and forest science are eventoday constantly striving to find and produce plants with an increasedgrowth potential to feed an increasing world population and to guaranteea supply of reproducible raw materials. The robust level of research inthese fields of science indicates the level of importance leaders inevery geographic environment and climate around the world place onproviding sustainable sources of food, feed and energy.

Manipulation of crop performance has been accomplished conventionallyfor centuries through selection and plant breeding. The breeding processis, however, both time-consuming and labor-intensive. Furthermore,appropriate breeding programs must be specially designed for eachrelevant plant species.

On the other hand, great progress has been made in using moleculargenetic approaches to manipulate plants to provide better crops. Throughthe introduction and expression of recombinant nucleic acid molecules inplants, researchers are now poised to provide the community with plantspecies tailored to grow more efficiently and yield more product despitesuboptimal geographic and/or climatic environments. These new approacheshave the additional advantage of not being limited to one plant species,but instead being applicable to multiple different plant species (Zhanget al. (2004) Plant Physiol. 135:615; Zhang et al. (2001) Proc. Natl.Acad. Sci. USA 98:12832).

SUMMARY

This document provides methods and materials related to plants havingmodulated levels of tolerance to salinity and/or oxidative stress. Forexample, this document provides transgenic plants and plant cells havingincreased levels of tolerance to salinity and/or oxidative stress,nucleic acids used to generate transgenic plants and plant cells havingincreased levels of tolerance to salinity and/or oxidative stress, andmethods for making plants and plant cells having increased levels oftolerance to salinity and/or oxidative stress. Such plants and plantcells provide the opportunity to produce crops or plants under salineand/or oxidative stress conditions without stunted growth and diminishedyields. Increased levels of tolerance to salinity and/or oxidativestress may be useful to produce biomass which may be converted to aliquid fuel or other chemicals and/or to produce food and feed on landthat is currently marginally productive, resulting in an overallexpansion of arable land.

Methods of producing a plant and/or plant tissue are provided herein. Inone aspect, a method comprises growing a plant cell comprising anexogenous nucleic acid. The exogenous nucleic acid comprises aregulatory region operably linked to a nucleotide sequence encoding apolypeptide. The Hidden Markov Model (HMM) bit score of the amino acidsequence of the polypeptide is greater than about 30 using an HMMgenerated from the amino acid sequences depicted in one of FIGS. 1-6.The plant and/or plant tissue has a difference in the level of toleranceto salinity and/or oxidative stress as compared to the correspondinglevel in tolerance to salinity and/or oxidative stress of a controlplant that does not comprise the exogenous nucleic acid. In someembodiments the amino acid sequence of the polypeptide has an HMM bitscore greater than about 400 using an HMM generated from the amino acidsequences depicted in FIG. 1. In some embodiments the amino acidsequence of the polypeptide has an HMM bit score greater than about 30using an HMM generated from the amino acid sequences depicted in FIG. 2.In some embodiments the amino acid sequence of the polypeptide has anHMM bit score greater than about 120 using an HMM generated from theamino acid sequences depicted in FIG. 3. In some embodiments the aminoacid sequence of the polypeptide has an HMM bit score greater than about150 using an HMM generated from the amino acid sequences depicted inFIG. 4. In some embodiments the amino acid sequence of the polypeptidehas an HMM bit score greater than about 425 using an HMM generated fromthe amino acid sequences depicted in FIG. 5. In some embodiments theamino acid sequence of the polypeptide has an HMM bit score greater thanabout 550 using an HMM generated from the amino acid sequences depictedin FIG. 6.

In another aspect, a method comprises growing a plant cell comprising anexogenous nucleic acid. The exogenous nucleic acid comprises aregulatory region operably linked to a nucleotide sequence encoding apolypeptide having 85 percent or greater sequence identity to an aminoacid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 9, 11, 13, 14, 15,17, 19, 20, 22, 23, 24, 25, 27, 29, 30, 31, 33, 35, 36, 37, 38, 39, 41,42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 62, 63, 64, 66, 68, 69,71, 73, 74, 76, 78, 80, 81, 83, 84, 86, 88, 90, 91, 93, 94, 96, 98, 100,101, 102, 104, 106, 107, 109, 110, 112, 114, 116, 118, 119, 121, 122,123, 125, 126, 127, 128, 129, 130, 132, 134, 136, 138, 140, 141, 142,143, 144, 145, 147, 149, 151, 153, 154, 156, 158, 160, 162, 163, 165,166, 167, 168, and amino acid coordinates 1 to 135 of SEQ ID NO: 140. Aplant produced from the plant cell has a difference in the level oftolerance to salinity and/or oxidative stress as compared to thecorresponding level in a control plant that does not comprise theexogenous nucleic acid.

In another aspect, a method comprises growing a plant cell comprising anexogenous nucleic acid. The exogenous nucleic acid comprises aregulatory region operably linked to a nucleotide sequence having 85percent or greater sequence identity to at least a fragment of anucleotide sequence set forth in SEQ ID NOs. 1, 3, 5, 7, 10, 12, 16, 18,21, 26, 28, 32, 34, 40, 46, 48, 51, 53, 55, 57, 59, 61, 65, 67, 70, 72,75, 77, 79, 82, 85, 87, 89, 92, 95, 97, 99, 103, 105, 108, 111, 113,115, 117, 120, 124, 131, 133, 135, 137, 139, 146, 148, 150, 152, 155,157, 159, 161, and 164 and to a nucleotide sequence encoding any of theamino acid sequences set forth in the sequence listing. A plant and/orplant tissue produced from the plant cell has a difference in the levelof salinity and/or oxidative stress tolerance as compared to thecorresponding level in a control plant that does not comprise theexogenous nucleic acid.

Methods of modulating the level of salt tolerance and/or oxidativestress tolerance in a plant are provided herein. In one aspect, a methodcomprises introducing into a plant cell an exogenous nucleic acid, thatcomprises a regulatory region operably linked to a nucleotide sequenceencoding a polypeptide. The HMM bit score of the amino acid sequence ofthe polypeptide is greater than 30, using an HMM generated from theamino acid sequences depicted in one of FIGS. 1-6. A plant and/or planttissue produced from the plant cell has a difference in the level oftolerance to salinity and/or oxidative stress as compared to thecorresponding level in a control plant that does not comprise theexogenous nucleic acid.

In another aspect, a method comprises introducing into a plant cell anexogenous nucleic acid that comprises a regulatory region operablylinked to a nucleotide sequence encoding a polypeptide having 85%percent or greater sequence identity to an amino acid sequence set forthin SEQ ID NOs: 2, 4, 6, 8, 9, 11, 13, 14, 15, 17, 19, 20, 22, 23, 24,25, 27, 29, 30, 31, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 47, 49,50, 52, 54, 56, 58, 60, 62, 63, 64, 66, 68, 69, 71, 73, 74, 76, 78, 80,81, 83, 84, 86, 88, 90, 91, 93, 94, 96, 98, 100, 101, 102, 104, 106,107, 109, 110, 112, 114, 116, 118, 119, 121, 122, 123, 125, 126, 127,128, 129, 130, 132, 134, 136, 138, 140, 141, 142, 143, 144, 145, 147,149, 151, 153, 154, 156, 158, 160, 162, 163, 165, 166, 167, 168, andamino acid coordinates 1 to 135 of SEQ ID NO: 140. A plant and/or planttissue produced from the plant cell has a difference in the level oftolerance to salinity or oxidative stress as compared to thecorresponding level in a control plant that does not comprise theexogenous nucleic acid.

In some embodiments, the methods comprise introducing into the plantcell an exogenous nucleic acid encoding polypeptides selected from thegroup consisting of SEQ ID NOs: 43, 44, 45, 86, 140, 141, 142, 143, 144,and amino acid coordinates 1 to 135 of SEQ ID NO: 140. A plant and/orplant tissue produced from the plant cell has a difference in the levelof tolerance to salinity as compared to the corresponding level in acontrol plant that does not comprise the exogenous nucleic acid. In someembodiments, the methods comprise introducing into the plant cell anexogenous nucleic acid encoding polypeptides selected from the groupconsisting of SEQ ID NO: 136, and 141, and a plant and/or plant tissueproduced from the plant cell has a difference in the level of toleranceto oxidative stress as compared to the corresponding level in a controlplant that does not comprise the exogenous nucleic acid.

In another aspect, a method comprises introducing into a plant cell anexogenous nucleic acid, that comprises a regulatory region operablylinked to a nucleotide sequence having 85 percent or greater sequenceidentity to a nucleotide sequence set forth in SEQ ID NOs: 1, 3, 5, 7,10, 12, 16, 18, 21, 26, 28, 32, 34, 40, 46, 48, 51, 53, 55, 57, 59, 61,65, 67, 70, 72, 75, 77, 79, 82, 85, 87, 89, 92, 95, 97, 99, 103, 105,108, 111, 113, 115, 117, 120, 124, 131, 133, 135, 137, 139, 146, 148,150, 152, 155, 157, 159, 161, and 164 and to a nucleotide sequenceencoding any of the amino acid sequences set forth in the sequencelisting. A plant and/or plant tissue produced from the plant cell has adifference in the level of tolerance to salinity or oxidative stress ascompared to the corresponding level in a control plant that does notcomprise the exogenous nucleic acid.

Plant cells comprising an exogenous nucleic acid are provided herein. Inone aspect, the exogenous nucleic acid comprises a regulatory regionoperably linked to a nucleotide sequence encoding a polypeptide. The HMMbit score of the amino acid sequence of the polypeptide is greater than30, using an HMM based on the amino acid sequences depicted in one ofFIGS. 1-6. The plant and/or plant tissue has a difference in the levelof tolerance to salinity or oxidative stress as compared to thecorresponding level in a control plant that does not comprise theexogenous nucleic acid. In another aspect, the exogenous nucleic acidcomprises a regulatory region operably linked to a nucleotide sequenceencoding a polypeptide having 85 percent or greater sequence identity toan amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 9, 11, 13, 14, 15, 17, 19, 20, 22, 23, 24, 25, 27, 29, 30,31, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 47, 49, 50, 52, 54, 56,58, 60, 62, 63, 64, 66, 68, 69, 71, 73, 74, 76, 78, 80, 81, 83, 84, 86,88, 90, 91, 93, 94, 96, 98, 100, 101, 102, 104, 106, 107, 109, 110, 112,114, 116, 118, 119, 121, 122, 123, 125, 126, 127, 128, 129, 130, 132,134, 136, 138, 140, 141, 142, 143, 144, 145, 147, 149, 151, 153, 154,156, 158, 160, 162, 163, 165, 166, 167, 168, and amino acid coordinates1 to 135 of SEQ ID NO: 140 A plant and/or plant tissue produced from theplant cell has a difference in the level of tolerance to salinity oroxidative stress as compared to the corresponding level in a controlplant that does not comprise the exogenous nucleic acid. In anotheraspect, the exogenous nucleic acid comprises a regulatory regionoperably linked to a nucleotide sequence having 85 percent or greatersequence identity to at least a fragment of a nucleotide sequenceselected from the group consisting of SEQ ID Nos. 1, 3, 5, 7, 10, 12,16, 18, 21, 26, 28, 32, 34, 40, 46, 48, 51, 53, 55, 57, 59, 61, 65, 67,70, 72, 75, 77, 79, 82, 85, 87, 89, 92, 95, 97, 99, 103, 105, 108, 111,113, 115, 117, 120, 124, 131, 133, 135, 137, 139, 146, 148, 150, 152,155, 157, 159, 161, and 164, and to a nucleotide sequence encoding anyof the amino acid sequences set forth in the sequence listing. A plantand/or plant tissue produced from the plant cell has a difference in thelevel of tolerance to salinity or oxidative stress as compared to thecorresponding level in a control plant that does not comprise theexogenous nucleic acid. A transgenic plant comprising such a plant cellis also provided. In some embodiments, the transgenic plant is a memberof a species selected from the group consisting of Panicum virgatum(switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthusgiganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera(poplar), Zea mays (corn), Glycine max (soybean), Brassica napus(canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryzasativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa),Beta vulgaris (sugarbeet), or Pennisetum glaucum (pearl millet). Someembodiments are related to products comprising seed or vegetative tissuefrom transgenic plants as described above. Some embodiments relate tofood or feed products from transgenic plants as described above.

In another aspect, an isolated nucleic acid comprises a nucleotidesequence encoding a polypeptide having 80% or greater sequence identityto the amino acid sequence set forth in SEQ ID Nos. 2, 4, 6, 22, 27, 29,49, 52, 54, 56, 60, 62, 68, 76, 83, 88, 90, 96, 98, 104, 106, 112, 114,132, 134, 149, 151, or 160.

In another aspect, methods of identifying a genetic polymorphismassociated with variation in the level of salinity and/or oxidativestress tolerance are provided. The methods include providing apopulation of plants, and determining whether one or more geneticpolymorphisms in the population are genetically linked to the locus fora polypeptide selected from the group consisting of the polypeptidesdepicted in FIGS. 1-6 and functional homologs thereof. The correlationbetween variation in the level of salinity tolerance and/or oxidativestress tolerance in plants and/or plant tissues of the population andthe presence of the one or more polymorphisms in plants of thepopulation is measured, thereby permitting identification of whether ornot the one or more polymorphisms are associated with such variation.

In another aspect, methods of making a plant line is provided. Themethods include determining whether one or more genetic polymorphisms ina population of plants is associated with the locus for a polypeptideselected from the group consisting of the polypeptides depicted in FIGS.1-6 and functional homologs thereof, identifying one or more plants inthe population in which the presence of at least one allele at the oneor more polymorphisms is associated with variation in salt tolerance oroxidative stress tolerance, crossing each of the one or more identifiedplants with itself or a different plant to produce seed, crossing atleast one progeny plant grown from said seed with itself or a differentplant, and repeating the crossing steps for an additional 0-5generations to make the plant line. The at least one allele will bepresent in the plant line. The method of making a plant line may beapplied, for example, to a population of switchgrass plants.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an alignment of amino acid sequences of homologues of(ME08768; SEQ ID NO: 86). In all the alignment Figures shown herein, adash in an aligned sequence represents a gap, i.e., a lack of an aminoacid at that position. Identical amino acids or conserved amino acidsubstitutions among aligned sequences are identified by boxes. FIG. 1and the other alignment Figures provided herein were generated using theprogram MUSCLE version 3.52

FIG. 2 is an alignment of amino acid sequences of homologues of ME06748(SEQ ID NO: 41).

FIG. 3 is an alignment of amino acid sequences of homologues of ME19173(SEQ ID NO: 109).

FIG. 4 is an alignment of amino acid sequences of homologues of ME02064C(SEQ ID NO: 140).

FIG. 5 is an alignment of amino acid sequences of homologues of CeresClone ID No. 1792354 (SEQ ID NO:2).

FIG. 6 is an alignment of amino acid sequences of homologues of CeresClone ID No. 56784328 (SEQ ID NO: 35).

DETAILED DESCRIPTION

The invention features methods and materials related to modulatingsalinity tolerance and/or oxidative stress tolerance levels in plantsand/or plant tissues. In some embodiments, the plants may also haveincreased biomass and/or yield. The methods can include transforming aplant cell with a nucleic acid encoding a salinity and/or oxidativestress tolerance-modulating polypeptide, wherein expression of thepolypeptide results in a modulated level of salinity tolerance and/oroxidative stress tolerance. Plant cells produced using such methods canbe grown to produce plants having an increased salinity tolerance,oxidative stress tolerance, and/or biomass, in comparison to wild typeplants grown under the same conditions. Such plants, and the seeds ofsuch plants, may be used to produce, for example, yield and/or biomassutilized for biofuel production, such as, but not limited to, ethanoland butanol.

I. DEFINITIONS

“Amino acid” refers to one of the twenty biologically occurring aminoacids and to synthetic amino acids, including D/L optical isomers.

“Cell type-preferential promoter” or “tissue-preferential promoter”refers to a promoter that drives expression preferentially in a targetcell type or tissue, respectively, but may also lead to sometranscription in other cell types or tissues as well.

“Control plant” refers to a plant that does not contain the exogenousnucleic acid present in a transgenic plant of interest, but otherwisehas the same or similar genetic background as such a transgenic plant. Asuitable control plant can be a non-transgenic wild type plant, anon-transgenic segregant from a transformation experiment, or atransgenic plant that contains an exogenous nucleic acid other than theexogenous nucleic acid of interest.

“Domains” are groups of substantially contiguous amino acids in apolypeptide that can be used to characterize protein families and/orparts of proteins. Such domains have a “fingerprint” or “signature” thatcan comprise conserved primary sequence, secondary structure, and/orthree-dimensional conformation. Generally, domains are correlated withspecific in vitro and/or in vivo activities. A domain can have a lengthof from 10 amino acids to 400 amino acids, e.g., 10 to 50 amino acids,or 25 to 100 amino acids, or 35 to 65 amino acids, or 35 to 55 aminoacids, or 45 to 60 amino acids, or 200 to 300 amino acids, or 300 to 400amino acids.

“Down-regulation” refers to regulation that decreases production ofexpression products (mRNA, polypeptide, or both) relative to basal ornative states.

“Exogenous” with respect to a nucleic acid indicates that the nucleicacid is part of a recombinant nucleic acid construct, or is not in itsnatural environment. For example, an exogenous nucleic acid can be asequence from one species introduced into another species, i.e., aheterologous nucleic acid. Typically, such an exogenous nucleic acid isintroduced into the other species via a recombinant nucleic acidconstruct. An exogenous nucleic acid can also be a sequence that isnative to an organism and that has been reintroduced into cells of thatorganism. An exogenous nucleic acid that includes a native sequence canoften be distinguished from the naturally occurring sequence by thepresence of non-natural sequences linked to the exogenous nucleic acid,e.g., non-native regulatory sequences flanking a native sequence in arecombinant nucleic acid construct. In addition, stably transformedexogenous nucleic acids typically are integrated at positions other thanthe position where the native sequence is found. It will be appreciatedthat an exogenous nucleic acid may have been introduced into aprogenitor and not into the cell under consideration. For example, atransgenic plant containing an exogenous nucleic acid can be the progenyof a cross between a stably transformed plant and a non-transgenicplant. Such progeny are considered to contain the exogenous nucleicacid.

“Expression” refers to the process of converting genetic information ofa polynucleotide into RNA through transcription, which is catalyzed byan enzyme, RNA polymerase, and into protein, through translation of mRNAon ribosomes.

“Heterologous polypeptide” as used herein refers to a polypeptide thatis not a naturally occurring polypeptide in a plant cell, e.g., atransgenic Panicum virgatum plant transformed with and expressing thecoding sequence for a nitrogen transporter polypeptide from a Zea maysplant.

“Isolated nucleic acid” as used herein includes a naturally-occurringnucleic acid, provided one or both of the sequences immediately flankingthat nucleic acid in its naturally-occurring genome is removed orabsent. Thus, an isolated nucleic acid includes, without limitation, anucleic acid that exists as a purified molecule or a nucleic acidmolecule that is incorporated into a vector or a virus. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries, genomic libraries, or gel slices containing agenomic DNA restriction digest, is not to be considered an isolatednucleic acid.

“Modulation” of the level of a compound or constituent refers to thechange in the level of the indicated compound or constituent that isobserved as a result of expression of, or transcription from, anexogenous nucleic acid in a plant cell. The change in level is measuredrelative to the corresponding level in control plants.

“Nucleic acid” and “polynucleotide” are used interchangeably herein, andrefer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA,and DNA or RNA containing nucleic acid analogs. Polynucleotides can haveany three-dimensional structure. A nucleic acid can be double-strandedor single-stranded (i.e., a sense strand or an antisense strand).Non-limiting examples of polynucleotides include genes, gene fragments,exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, nucleic acid probes and nucleic acid primers. Apolynucleotide may contain unconventional or modified nucleotides.

“Operably linked” refers to the positioning of a regulatory region and asequence to be transcribed in a nucleic acid so that the regulatoryregion is effective for regulating transcription or translation of thesequence. For example, to operably link a coding sequence and aregulatory region, the translation initiation site of the translationalreading frame of the coding sequence is typically positioned between oneand about fifty nucleotides downstream of the regulatory region. Aregulatory region can, however, be positioned as much as about 5,000nucleotides upstream of the translation initiation site, or about 2,000nucleotides upstream of the transcription start site.

“Polypeptide” as used herein refers to a compound of two or more subunitamino acids, amino acid analogs, or other peptidomimetics, regardless ofpost-translational modification, e.g., phosphorylation or glycosylation.The subunits may be linked by peptide bonds or other bonds such as, forexample, ester or ether bonds. Full-length polypeptides, truncatedpolypeptides, point mutants, insertion mutants, splice variants,chimeric proteins, and fragments thereof are encompassed by thisdefinition.

“Progeny” includes descendants of a particular plant or plant line.Progeny of an instant plant include seeds formed on F₁, F₂, F₃, F₄, F₅,F₆ and subsequent generation plants, or seeds formed on BC₁, BC₂, BC₃,and subsequent generation plants, or seeds formed on F₁BC₁, F₁BC₂,F₁BC₃, and subsequent generation plants. The designation F₁ refers tothe progeny of a cross between two parents that are geneticallydistinct. The designations F₂, F₃, F₄, F₅ and F₆ refer to subsequentgenerations of self- or sib-pollinated progeny of an F₁ plant.

“Regulatory region” refers to a nucleic acid having nucleotide sequencesthat influence transcription or translation initiation and rate, andstability and/or mobility of a transcription or translation product.Regulatory regions include, without limitation, promoter sequences,enhancer sequences, response elements, protein recognition sites,inducible elements, protein binding sequences, 5′ and 3′ untranslatedregions (UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, introns, and combinations thereof. Aregulatory region typically comprises at least a core (basal) promoter.A regulatory region also may include at least one control element, suchas an enhancer sequence, an upstream element or an upstream activationregion (UAR). For example, a suitable enhancer is a cis-regulatoryelement (−212 to −154) from the upstream region of the octopine synthase(ocs) gene. Fromm et al., The Plant Cell, 1:977-984 (1989).

“Up-regulation” refers to regulation that increases the level of anexpression product (mRNA, polypeptide, or both) relative to basal ornative states.

“Vector” refers to a replicon, such as a plasmid, phage, or cosmid, intowhich another DNA segment may be inserted so as to bring about thereplication of the inserted segment. Generally, a vector is capable ofreplication when associated with the proper control elements. The term“vector” includes cloning and expression vectors, as well as viralvectors and integrating vectors. An “expression vector” is a vector thatincludes a regulatory region.

Oxidative stress: Plant species vary in their capacity to tolerateROS/ROI/AOS. “Oxidative stress” can be defined as the set ofenvironmental conditions under which a plant will begin to suffer theeffects of elevated ROS/ROI/AOS concentration, such as decreases inenzymatic activity, DNA breakage, DNA-protein crosslinking, necrosis andstunted growth. For these reasons, plants experiencing oxidative stresstypically exhibit a significant reduction in biomass and/or yield.

Elevated oxidative stress may be caused by natural, geological processesand by human activities, such as pollution. Since plant species vary intheir capacity to tolerate oxidative stress, the precise environmentalconditions that cause stress cannot be generalized. However, underoxidative stress conditions, oxidative stress tolerant plants producehigher biomass, yield and survivorship than plants that are notoxidative stress tolerant. Differences in physical appearance, recoveryand yield can be quantified

Photosynthetic efficiency: photosynthetic efficiency, or electrontransport via photosystem II, is estimated by the relationship betweenFm, the maximum fluorescence signal and the variable fluorescence, Fv. Areduction in the optimum quantum yield (Fv/Fm) indicates stress and canbe used to monitor the performance of transgenic plants compared tonon-transgenic plants under salt or oxidative stress conditions.

Salicylic Acid Growth Index (SAGI): Photosynthetic efficiency x seedlingarea.

Salt growth index (SGI): Photosynthetic efficiency x seedling area(under salinity stress condition).

Salinity: Plant species vary in their capacity to tolerate salinity.“Salinity” can be defined as the set of environmental conditions underwhich a plant will begin to suffer the effects of elevated saltconcentration, such as ion imbalance, decreased stomatal conductance,decreased photosynthesis, decreased growth rate, increased cell death,loss of turgor (wilting), or ovule abortion. For these reasons, plantsexperiencing salinity stress typically exhibit a significant reductionin biomass and/or yield.

Elevated salinity may be caused by natural, geological processes and byhuman activities, such as pollution. Since plant species vary in theircapacity to tolerate salinity, the precise environmental conditions thatcause stress cannot be generalized. However, under saline conditions,salinity tolerant plants produce higher biomass, yield and survivorshipthan plants that are not saline tolerant. Differences in physicalappearance, recovery and yield can be quantified.

Elevated salinity may be caused by natural, geological processes and byhuman activities, such as irrigation. Since plant species vary in theircapacity to tolerate water deficit, the precise environmental saltconditions that cause stress cannot be generalized. However, undersaline conditions, salt tolerant plants produce higher biomass, yieldand survivorship than plants that are not salt tolerant. Differences inphysical appearance, recovery and yield can be quantified andstatistically analyzed using well known measurement and analysismethods.

II. POLYPEPTIDES

Polypeptides described herein include salinity tolerance and/oroxidative stress tolerance-modulating polypeptides. Salinity toleranceand/or oxidative stress tolerance-modulating polypeptides can beeffective to modulate salinity tolerance and/or oxidative stresstolerance levels when expressed in a plant or plant cell. Suchpolypeptides typically contain at least one domain indicative ofsalinity tolerance and/or oxidative stress tolerance-modulatingpolypeptides, as described in more detail herein. Salinity toleranceand/or oxidative stress tolerance-modulating polypeptides typically havean HMM bit score that is greater than 30, as described in more detailherein. In some embodiments, salinity tolerance and/or oxidative stresstolerance-modulating polypeptides have greater than 85% identity to SEQID NOs: 2, 4, 6, 8, 9, 11, 13, 14, 15, 17, 19, 20, 22, 23, 24, 25, 27,29, 30, 31, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 47, 49, 50, 52,54, 56, 58, 60, 62, 63, 64, 66, 68, 69, 71, 73, 74, 76, 78, 80, 81, 83,84, 86, 88, 90, 91, 93, 94, 96, 98, 100, 101, 102, 104, 106, 107, 109,110, 112, 114, 116, 118, 119, 121, 122, 123, 125, 126, 127, 128, 129,130, 132, 134, 136, 138, 140, 141, 142, 143, 144, 145, 147, 149, 151,153, 154, 156, 158, 160, 162, 163, 165, 166, 167, 168, and amino acidcoordinates 1 to 135 of SEQ ID NO: 140 as described in more detailherein.

A. Domains Indicative of Salinity Tolerance and/or Oxidative StressTolerance—Modulating Polypeptides

A salinity tolerance and/or oxidative stress tolerance-modulatingpolypeptide can contain an IQ calmodulin-binding motif domain, which ispredicted to be characteristic of an salinity tolerance and/or oxidativestress tolerance-modulating polypeptide. Calmodulin (CaM) is recognizedas a major calcium sensor and orchestrator of regulatory events throughits interaction with a diverse group of cellular proteins. Three classesof recognition motifs exist for many of the known CaM binding proteins;the IQ motif as a consensus for Ca2⁺-independent binding and two relatedmotifs for Ca2⁺-dependent binding, termed 18-14 and 1-5-10 based on theposition of conserved hydrophobic residues PUBMED:9141499.

For example, the regulatory domain of scallop myosin is a three-chainprotein complex that switches on this motor in response to Ca2⁺ binding.Side-chain interactions link the two light chains in tandem to adjacentsegments of the heavy chain bearing the IQ-sequence motif. TheCa2⁺-binding site is a novel EF-hand motif on the essential light chainand is stabilized by linkages involving the heavy chain and both lightchains, accounting for the requirement of all three chains for Ca2⁺binding and regulation in the intact myosin molecule PUB MED:8127365.

For example, SEQ ID NO:86 sets forth the amino acid sequence of anArabidopsis clone, identified herein as Ceres SEEDLINE ID no. ME08768,that is predicted to encode a polypeptide containing a IQcalmodulin-binding motif domain from residues 116-136.

In some embodiments, a salinity tolerance and/or oxidative stresstolerance-modulating polypeptide is truncated at the amino- orcarboxy-terminal end of a naturally occurring polypeptide. A truncatedpolypeptide may retain certain domains of the naturally occurringpolypeptide while lacking others. Thus, length variants that are up to 5amino acids shorter or longer typically exhibit the salinity toleranceand/or oxidative stress tolerance-modulating activity of a truncatedpolypeptide. In some embodiments, a truncated polypeptide is a dominantnegative polypeptide. SEQ ID NO: 138 sets forth the amino sequence of asalinity tolerance and/or oxidative stress tolerance-modulatingpolypeptide that is truncated at the 5′ end relative to the naturallyoccurring polypeptide. Expression in a plant of such a truncatedpolypeptide confers a difference in the level of salinity toleranceand/or oxidative stress tolerance in a plant and/or plant tissue ascompared to the corresponding level a control plant and/or tissuethereof that does not comprise the truncation.

B. Functional Homologs Identified by Reciprocal BLAST

In some embodiments, one or more functional homologs of a referencesalinity tolerance and/or oxidative stress tolerance-modulatingpolypeptide defined by one or more of the pfam descriptions indicatedabove are suitable for use as salinity tolerance and/or oxidative stresstolerance-modulating polypeptides. A functional homolog is a polypeptidethat has sequence similarity to a reference polypeptide, and thatcarries out one or more of the biochemical or physiological function(s)of the reference polypeptide. A functional homolog and the referencepolypeptide may be natural occurring polypeptides, and the sequencesimilarity may be due to convergent or divergent evolutionary events. Assuch, functional homologs are sometimes designated in the literature ashomologs, or orthologs, or paralogs. Variants of a naturally occurringfunctional homolog, such as polypeptides encoded by mutants of a wildtype coding sequence, may themselves be functional homologs. Functionalhomologs can also be created via site-directed mutagenesis of the codingsequence for a salinity tolerance and/or oxidative stresstolerance-modulating polypeptide, or by combining domains from thecoding sequences for different naturally-occurring salinity toleranceand/or oxidative stress tolerance-modulating polypeptides (“domainswapping”). The term “functional homolog” is sometimes applied to thenucleic acid that encodes a functionally homologous polypeptide.

Functional homologs can be identified by analysis of nucleotide andpolypeptide sequence alignments. For example, performing a query on adatabase of nucleotide or polypeptide sequences can identify homologs ofsalinity tolerance and/or oxidative stress tolerance-modulatingpolypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, orPSI-BLAST analysis of nonredundant databases using a salinity toleranceand/or oxidative stress tolerance-modulating polypeptide amino acidsequence as the reference sequence. Amino acid sequence is, in someinstances, deduced from the nucleotide sequence. Those polypeptides inthe database that have greater than 40% sequence identity are candidatesfor further evaluation for suitability as a salinity tolerance and/oroxidative stress tolerance-modulating polypeptide. Amino acid sequencesimilarity allows for conservative amino acid substitutions, such assubstitution of one hydrophobic residue for another or substitution ofone polar residue for another. If desired, manual inspection of suchcandidates can be carried out in order to narrow the number ofcandidates to be further evaluated. Manual inspection can be performedby selecting those candidates that appear to have domains present insalinity tolerance and/or oxidative stress tolerance-modulatingpolypeptides, e.g., conserved functional domains.

Conserved regions can be identified by locating a region within theprimary amino acid sequence of a salinity tolerance and/or oxidativestress tolerance-modulating polypeptide that is a repeated sequence,forms some secondary structure (e.g., helices and beta sheets),establishes positively or negatively charged domains, or represents aprotein motif or domain. See, e.g., the Pfam web site describingconsensus sequences for a variety of protein motifs and domains on theWorld Wide Web at sanger.ac.uk/Software/Pfam/and pfam.janelia.org/. Adescription of the information included at the Pfam database isdescribed in Sonnhammer et al., Nucl. Acids Res., 26:320-322 (1998);Sonnhammer et al., Proteins, 28:405-420 (1997); and Bateman et al.,Nucl. Acids Res., 27:260-262 (1999). Conserved regions also can bedetermined by aligning sequences of the same or related polypeptidesfrom closely related species. Closely related species preferably arefrom the same family. In some embodiments, alignment of sequences fromtwo different species is adequate.

Typically, polypeptides that exhibit at least about 40% amino acidsequence identity are useful to identify conserved regions. Conservedregions of related polypeptides exhibit at least 45% amino acid sequenceidentity (e.g., at least 50%, at least 60%, at least 70%, at least 80%,or at least 90% amino acid sequence identity). In some embodiments, aconserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acidsequence identity.

Amino acid sequences of functional homologs of the polypeptide set forthin SEQ ID NO: 86 are provided in FIG. 1 and in the Sequence Listing.Such functional homologs include (SEQ ID NO: 88, 90, 91, 93, 94, 96, 98,100, 101, 102, 104, 106, and 107). In some cases, a functional homologof SEQ ID NO: 86 has an amino acid sequence with at least 50% sequenceidentity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence setforth in SEQ ID NO: 86.

Amino acid sequences of functional homologs of the polypeptide set forthin SEQ ID NO: 41 are provided in FIG. 2. Such functional homologsinclude (SEQ ID NO: 42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 62,63, 64, 66, 68, 69, 71, 73, 74, 76, 78, 80, 81, 83, and 84). In somecases, a functional homolog of SEQ ID NO: 41 has an amino acid sequencewith at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to theamino acid sequence set forth in SEQ ID NO: 41.

Amino acid sequences of functional homologs of the polypeptide set forthin SEQ ID NO: 109 are provided in FIG. 3. Such functional homologsinclude (SEQ ID NO: 110, 112, 114, 116, 118, 119, 121, 122, 123, 125,126, 127, 128, 129, 130, 132, and 134). In some cases, a functionalhomolog of SEQ ID NO: 109 has an amino acid sequence with at least 50%sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acidsequence set forth in SEQ ID NO: 109.

Amino acid sequences of functional homologs of the polypeptide set forthin SEQ ID NO:140 are provided in FIG. 4. Such functional homologsinclude (SEQ ID NO: 136, 138, 140, 141, 142, 143, 144, 145, 147, 149,151, 153, 154, 156, 158, 160, 162, 163, 165, 166, 167, and 168). In somecases, a functional homolog of SEQ ID NO: 140 has an amino acid sequencewith at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to theamino acid sequence set forth in SEQ ID NO: 140.

Amino acid sequences of functional homologs of the polypeptide set forthin SEQ ID NO: 2 are provided in FIG. 5. Such functional homologs include(SEQ ID NO: 2, 4, 6, 8, 9, 11, 13, 14, 15, 17, 19, 20, 22, 23, 24, 25,27, 29, 30, 31, and 33). In some cases, a functional homolog of SEQ IDNO: 2 has an amino acid sequence with at least 50% sequence identity,e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, or 99% sequence identity, to the amino acid sequence set forth inSEQ ID NO: 2.

Amino acid sequences of functional homologs of the polypeptide set forthin SEQ ID NO: 35 are provided in FIG. 6. Such functional homologsinclude (SEQ ID NO: 35, 36, 37, 38, and 39). In some cases, a functionalhomolog of SEQ ID NO: 35 has an amino acid sequence with at least 50%sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acidsequence set forth in SEQ ID NO: 35.

The identification of conserved regions in a salinity tolerance and/oroxidative stress tolerance-modulating polypeptide facilitates productionof variants of salinity tolerance and/or oxidative stresstolerance-modulating polypeptides. Variants of salinity tolerance and/oroxidative stress tolerance-modulating polypeptides typically have 10 orfewer conservative amino acid substitutions within the primary aminoacid sequence, e.g., 7 or fewer conservative amino acid substitutions, 5or fewer conservative amino acid substitutions, or between 1 and 5conservative substitutions. A useful variant polypeptide can beconstructed based on one of the alignments set forth in FIGS. 1 thru 6.Such a polypeptide includes the conserved regions, arranged in the orderdepicted in the Figure from amino-terminal end to carboxy-terminal end.Such a polypeptide may also include zero, one, or more than one aminoacid in positions marked by dashes. When no amino acids are present atpositions marked by dashes, the length of such a polypeptide is the sumof the amino acid residues in all conserved regions. When amino acidsare present at all positions marked by dashes, such a polypeptide has alength that is the sum of the amino acid residues in all conservedregions and all dashes.

C. Functional Homologues Identified by HMM

In some embodiments, useful salinity and/or oxidative stresstolerance-modulating polypeptides include those that fit a Hidden MarkovModel based on the polypeptides set forth in any one of FIGS. 1-6. AHidden Markov Model (HMM) is a statistical model of a consensus sequencefor a group of functional homologs. See, Durbin et al., BiologicalSequence Analysis: Probabilistic Models of Proteins and Nucleic Acids,Cambridge University Press, Cambridge, UK (1998). An HMM is generated bythe program HMMER 2.3.2 with default program parameters, using thesequences of the group of functional homologs as input. The multiplesequence alignment is generated by ProbCons (Do et al., Genome Res.,15(2):330-40 (2005)) version 1.11 using a set of default parameters: -c,--consistency REPS of 2; -ir, --iterative-refinement REPS of 100; -pre,--pre-training REPS of 0. ProbCons is a public domain software programprovided by Stanford University.

The default parameters for building an HMM (hmmbuild) are as follows:the default “architecture prior” (archpri) used by MAP architectureconstruction is 0.85, and the default cutoff threshold (idlevel) used todetermine the effective sequence number is 0.62. HMMER 2.3.2 wasreleased Oct. 3, 2003 under a GNU general public license, and isavailable from various sources on the World Wide Web. Hmmbuild outputsthe model as a text file.

The HMM for a group of functional homologs can be used to determine thelikelihood that a candidate salinity tolerance and/or oxidative stresstolerance-modulating polypeptide sequence is a better fit to thatparticular HMM than to a null HMM generated using a group of sequencesthat are not structurally or functionally related. The likelihood that asubject polypeptide sequence is a better fit to an HMM than to a nullHMM is indicated by the HMM bit score, a number generated when thecandidate sequence is fitted to the HMM profile using the HMMERhmmsearch program. The following default parameters are used whenrunning hmmsearch: the default E-value cutoff (E) is 10.0, the defaultbit score cutoff (T) is negative infinity, the default number ofsequences in a database (Z) is the real number of sequences in thedatabase, the default E-value cutoff for the per-domain ranked hit list(domE) is infinity, and the default bit score cutoff for the per-domainranked hit list (domT) is negative infinity. A high HMM bit scoreindicates a greater likelihood that the subject sequence carries out oneor more of the biochemical or physiological function(s) of thepolypeptides used to generate the HMM. A high HMM bit score is at least20, and often is higher.

As those of skill in the art would appreciate, the HMM scores providedin the sequence listing are merely exemplary. Since multiple sequencealignment algorithms, such as ProbCons, can only generate near-optimalresults, slight variations of the model can arise due to factors such asthe order in which sequences are processed for alignment. Nevertheless,HMM score variability is minor, and so the HMM scores in the sequencelisting are representative of models made with the respective sequences.

The salinity and/or oxidative stress-modulating polypeptides discussedbelow fit the indicated HMM with an HMM bit score greater than 20 (e.g.,greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or500). In some embodiments, the HMM bit score of a salinity and/oroxidative stress-modulating polypeptide discussed below is about 50%,60%, 70%, 80%, 90%, or 95% of the HMM bit score of a functional homologprovided in the Sequence Listing. In some embodiments, a salinity and/oroxidative stress-modulating polypeptide discussed below fits theindicated HMM with an HMM bit score greater than 20, and has a domainindicative of an salinity and/or oxidative stress-modulatingpolypeptide. In some embodiments, a salinity and/or oxidativestress-modulating polypeptide discussed below fits the indicated HMMwith an HMM bit score greater than 20, and has 85% or greater sequenceidentity (e.g., 75%, 80%, 85%, 90%, 95%, or 100% sequence identity) toan amino acid sequence shown in any one of FIGS. 1 thru 6 or to an aminoacid sequence correlated in the Sequence Listing to a any one of FIGS. 1thru 6._.

In the Sequence Listing polypeptides are provided that have HMM bitscores greater than 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, or 1100, when fitted to an HMM generated from theamino acid sequences set forth in FIG. 1. Such polypeptides includeCeres SEEDLINE ID no. ME08768, Ceres CLONE ID no. 1943807, Ceres ANNOTID no. 1471392, Public GI ID no. 6715635, Ceres CLONE ID no. 910109,Public GI ID no. 115474509, Ceres CLONE ID no. 1780908, Ceres ANNOT IDno. 1520883, Ceres CLONE ID no. 148018, Public GI ID no. 18378797,Public GI ID no. 21553500, Ceres ANNOT ID no. 1444522, Ceres ANNOT IDno. 146751, and Public GI ID no. 125559938 (SEQ ID NO: 86, 88, 90, 91,93, 94, 96, 98, 100, 101, 102, 104, 106, and 107)

In the Sequence Listing polypeptides are provided that have HMM bitscores greater than 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 when fitted to anHMM generated from the amino acid sequences set forth in FIG. 2. Suchpolypeptides include Ceres SEEDLINE ID no. ME06748, Ceres SEEDLINE IDno. ME20711, Ceres SEEDLINE ID no. ME18973, Ceres SEEDLINE ID no.ME08732, Ceres SEEDLINE ID no. ME19657, Ceres CLONE ID no. 835818, CeresCLONE ID no. 1796745, Public GI ID no. 125543896, Ceres ANNOT ID no.1483984, Ceres CLONE ID no. 1924654, Ceres ANNOT ID no. 1468861, CeresCLONE ID no. 1641776, Ceres ANNOT ID no. 1438750, Ceres ANNOT ID no.1447395, Public GI ID no. 79482785, Public GI ID no. 3292832, CeresCLONE ID no. 1559074, Ceres CLONE ID no. 1726548, Public GI ID no.115459996, Ceres CLONE ID no. 697034, Ceres CLONE ID no. 353438, PublicGI ID no. 125593074, Ceres CLONE ID no. 1920115, Ceres CLONE ID no.21821, Ceres CLONE ID no. 560066, Public GI ID no. 115453071, CeresCLONE ID no. 1968211, and Public GI ID no. 116310011_(SEQ ID NO: 42, 43,44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 62, 63, 64, 66, 68, 69, 71, 73,74, 76, 78, 80, 81, 83, and 84).

In the Sequence Listing polypeptides are provided that have HMM bitscores greater than 120, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200when fitted to an HMM generated from the amino acid sequences set forthin FIG. 3. Such polypeptides include Ceres SEEDLINE ID no. ME19173,Public GI ID no. 115435054, Ceres CLONE ID no. 1847857, Ceres ANNOT IDno. 1455219, Ceres CLONE ID no. 352452, Ceres CLONE ID no. 787908, CeresLOCUS ID no. Os01m00929_AP002743, Ceres CLONE ID no. 246398, Public GIID no. 125527441, Public GI ID no. 125595056, Ceres CLONE ID no. 236071,Public GI ID no. 125524760, Public GI ID no. 125569365, Public GI ID no.115439499, Public GI ID no. 15225258, Public GI ID no. 115465173, CeresANNOT ID no. 1477059, and Ceres ANNOT ID no. 1530547 (SEQ ID NO: 110,112, 114, 116, 118, 119, 121, 122, 123, 125, 126, 127, 128, 129, 130,132, and 134).

In the Sequence Listing polypeptides are provided that have HMM bitscores greater than 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, or 1350 when fitted to an HMM generated from the amino acidsequences set forth in FIG. 4. Such polypeptides include Ceres SEEDLINEID no. ME24091, Ceres CLONE ID no. 375578, Ceres CLONE ID no. 375578,Ceres SEEDLINE ID no. ME10681, Ceres SEEDLINE ID no. ME03140, CeresSEEDLINE ID no. ME24076, Ceres SEEDLINE ID no. ME24217, Public GI ID no.115440873, Ceres CLONE ID no. 826796, Ceres ANNOT ID no. 1465047, CeresCLONE ID no. 1919901, Ceres CLONE ID no. 520008, Public GI ID no.7413581, Ceres CLONE ID no. 228069, Ceres CLONE ID no. 467508, CeresCLONE ID no. 1829581, Ceres CLONE ID no. 229668, Public GI ID no.125550655, Ceres CLONE ID no. 106263, Public GI ID no. 15231175, PublicGI ID no. 145357576, and Public GI ID no. 125528277 (SEQ ID NO: 136,138, 140, 141, 142, 143, 144, 145, 147, 149, 151, 153, 154, 156, 158,160, 162, 163, 165, 166, 167, and 168).

In the Sequence Listing polypeptides are provided that have HMM bitscores greater than 425, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500, or 1550, when fitted to an HMM generated from the amino acidsequences set forth in FIG. 5. Such polypeptides include Ceres CLONE IDno. 1792354, Ceres CLONE ID no. 1925477, Ceres ANNOT ID no. 1521592,Ceres CLONE ID no. 463594, Public GI ID no. 22330633, Ceres CLONE ID no.345954, Ceres LOCUS ID no. Os01m05025_AP003288, GI ID no. 56784330,Public GI ID no. 125527495, Public GI ID no. 125553119, Ceres CLONE IDno. 236431, Ceres CLONE ID no. 908518, Public GI ID no. 115465121, CeresCLONE ID no. 1791910, Public GI ID no. 125595019, Public GI ID no.42568886, Public GI ID no. 2947062, Ceres ANNOT ID no. 1468228, CeresCLONE ID no. 1942388, Public GI ID no. 12324824, Public GI ID no.5882749, and Ceres CLONE ID no. 325403 (SEQ ID NO: 2, 4, 6, 8, 9, 11,13, 14, 15, 17, 19, 20, 22, 23, 24, 25, 27, 29, 30, 31, and 33).

In the Sequence Listing polypeptides are provided that have HMM bitscores greater than 550, 600, 650, or 700 when fitted to an HMMgenerated from the amino acid sequences set forth in FIG. 6. Suchpolypeptides include Ceres GI ID no. 56784328, Public GI ID no.56784330, Public GI ID no. 125528718, Public GI ID no. 125572975, andPublic GI ID no. 125528716 (SEQ ID NO: 35, 36, 37, 38, and 39).

D. Percent Identity

In some embodiments, a salinity and/or oxidative stresstolerance-modulating polypeptide has an amino acid sequence with atleast 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to one ofthe amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 9, 11, 13,14, 15, 17, 19, 20, 22, 23, 24, 25, 27, 29, 30, 31, 33, 35, 36, 37, 38,39, 41, 42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 62, 63, 64, 66,68, 69, 71, 73, 74, 76, 78, 80, 81, 83, 84, 86, 88, 90, 91, 93, 94, 96,98, 100, 101, 102, 104, 106, 107, 109, 110, 112, 114, 116, 118, 119,121, 122, 123, 125, 126, 127, 128, 129, 130, 132, 134, 136, 138, 140,141, 142, 143, 144, 145, 147, 149, 151, 153, 154, 156, 158, 160, 162,163, 165, 166, 167, 168, and amino acid coordinates 1 to 135 of SEQ IDNO: 140. Polypeptides having such a percent sequence identity often havea domain indicative of a salinity and/or oxidative stress-modulatingpolypeptide and/or have an HMM bit score that is greater than 20, asdiscussed above. Examples of amino acid sequences of salinity and/oroxidative stress tolerance-modulating polypeptides having at least 85%sequence identity to one of the amino acid sequences set forth in SEQ IDNOs: 2, 4, 6, 8, 9, 11, 13, 14, 15, 17, 19, 20, 22, 23, 24, 25, 27, 29,30, 31, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 47, 49, 50, 52, 54,56, 58, 60, 62, 63, 64, 66, 68, 69, 71, 73, 74, 76, 78, 80, 81, 83, 84,86, 88, 90, 91, 93, 94, 96, 98, 100, 101, 102, 104, 106, 107, 109, 110,112, 114, 116, 118, 119, 121, 122, 123, 125, 126, 127, 128, 129, 130,132, 134, 136, 138, 140, 141, 142, 143, 144, 145, 147, 149, 151, 153,154, 156, 158, 160, 162, 163, 165, 166, 167, 168, and amino acidcoordinates 1 to 135 of SEQ ID NO: 140 are provided in FIGS. 1-6.

“Percent sequence identity” refers to the degree of sequence identitybetween any given reference sequence, e.g., SEQ ID NOs: 2, 4, 6, 8, 9,11, 13, 14, 15, 17, 19, 20, 22, 23, 24, 25, 27, 29, 30, 31, 33, 35, 36,37, 38, 39, 41, 42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 62, 63,64, 66, 68, 69, 71, 73, 74, 76, 78, 80, 81, 83, 84, 86, 88, 90, 91, 93,94, 96, 98, 100, 101, 102, 104, 106, 107, 109, 110, 112, 114, 116, 118,119, 121, 122, 123, 125, 126, 127, 128, 129, 130, 132, 134, 136, 138,140, 141, 142, 143, 144, 145, 147, 149, 151, 153, 154, 156, 158, 160,162, 163, 165, 166, 167, 168, and amino acid coordinates 1 to 135 of SEQID NO: 140, and a candidate salinity and/or oxidative stress-modulatingsequence. A candidate sequence typically has a length that is from 80percent to 200 percent of the length of the reference sequence, e.g.,82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, 120, 130, 140,150, 160, 170, 180, 190, or 200 percent of the length of the referencesequence. A percent identity for any candidate nucleic acid orpolypeptide relative to a reference nucleic acid or polypeptide can bedetermined as follows. A reference sequence (e.g., a nucleic acidsequence or an amino acid sequence) is aligned to one or more candidatesequences using the computer program ClustalW (version 1.83, defaultparameters), which allows alignments of nucleic acid or polypeptidesequences to be carried out across their entire length (globalalignment). Chenna et al., Nucleic Acids Res., 31(13):3497-500 (2003).

ClustalW calculates the best match between a reference and one or morecandidate sequences, and aligns them so that identities, similaritiesand differences can be determined. Gaps of one or more residues can beinserted into a reference sequence, a candidate sequence, or both, tomaximize sequence alignments. For fast pairwise alignment of nucleicacid sequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For multiple alignment of nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The ClustalW output is asequence alignment that reflects the relationship between sequences.ClustalW can be run, for example, at the Baylor College of MedicineSearch Launcher site(searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at theEuropean Bioinformatics Institute site on the World Wide Web(ebi.ac.uk/clustalw).

To determine percent identity of a candidate nucleic acid or amino acidsequence to a reference sequence, the sequences are aligned usingClustalW, the number of identical matches in the alignment is divided bythe length of the reference sequence, and the result is multiplied by100. It is noted that the percent identity value can be rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded upto 78.2.

In some cases, a salinity and/or oxidative stress tolerance-modulatingpolypeptide has an amino acid sequence with at least 50% sequenceidentity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, or 99% sequence identity, to one or more of the aminoacid sequence set forth in SEQ ID NO: 86 Amino acid sequences ofpolypeptides having high sequence identity to the polypeptide set forthin SEQ ID NO: 86 are provided in the Sequence Listing. Such polypeptidesinclude SEQ ID NO: 88, 90, 91, 93, 94, 96, 98, 100, 101, 102, 104, 106,and 107.

In some cases, a salinity and/or oxidative stress tolerance-modulatingpolypeptide has an amino acid sequence with at least 50% sequenceidentity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence setforth in SEQ ID NO: 41. Amino acid sequences of polypeptides having highsequence identity to the polypeptide set forth in SEQ ID NO: 41 areprovided in the Sequence Listing. Such polypeptides include SEQ ID NO:42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 62, 63, 64, 66, 68, 69,71, 73, 74, 76, 78, 80, 81, 83, and 84.

In some cases, a salinity and/or oxidative stress-modulating polypeptidehas an amino acid sequence with at least 50% sequence identity, e.g.,50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or99% sequence identity, to the amino acid sequence set forth in SEQ IDNO: 109. Amino acid sequences of polypeptides having high sequenceidentity to the polypeptide set forth in SEQ ID NO: 109 are provided inthe Sequence Listing. Such polypeptides include SEQ ID NO: 110, 112,114, 116, 118, 119, 121, 122, 123, 125, 126, 127, 128, 129, 130, 132,and 134.

In some cases, a salinity and/or oxidative stress-modulating polypeptidehas an amino acid sequence with at least 50% sequence identity, e.g.,50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or99% sequence identity, to the amino acid sequence set forth in SEQ IDNO: 140. Amino acid sequences of polypeptides having high sequenceidentity to the polypeptide set forth in SEQ ID NO: 140 are provided inthe Sequence Listing. Such polypeptides include SEQ ID NO: 136, 138,141, 142, 143, 144, 145, 147, 149, 151, 153, 154, 156, 158, 160, 162,163, 165, 166, 167, and 168.

In some cases, a salinity and/or oxidative stress-modulating polypeptidehas an amino acid sequence with at least 50% sequence identity, e.g.,50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or99% sequence identity, to the amino acid sequence set forth in SEQ IDNO: 2. Amino acid sequences of polypeptides having high sequenceidentity to the polypeptide set forth in SEQ ID NO: 2 are provided inthe Sequence Listing. Such polypeptides include SEQ ID NO: 4, 6, 8, 9,11, 13, 14, 15, 17, 19, 20, 22, 23, 24, 25, 27, 29, 30, 31, and 33.

In some cases, a salinity and/or oxidative stress-modulating polypeptidehas an amino acid sequence with at least 50% sequence identity, e.g.,50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or99% sequence identity, to the amino acid sequence set forth in SEQ IDNO: 35. Amino acid sequences of polypeptides having high sequenceidentity to the polypeptide set forth in SEQ ID NO: 35 are provided inthe Sequence Listing. Such polypeptides include SEQ ID NO: 36, 37, 38,and 39.

E. Other Sequences

It should be appreciated that a salinity and/or oxidative stresstolerance-modulating polypeptide can include additional amino acids thatare not involved in salinity and/or oxidative stress tolerancemodulation, and thus such a polypeptide can be longer than wouldotherwise be the case. For example, a salinity and/or oxidativestress-tolerance modulating polypeptide can include a purification tag,a chloroplast transit peptide, an amyloplast transit peptide, amitochondrial transit peptide, or a leader sequence added to the aminoor carboxy terminus. In some embodiments, a salinity and/or oxidativestress-tolerance modulating polypeptide includes an amino acid sequencethat functions as a reporter, e.g., a green fluorescent protein oryellow fluorescent protein.

III. NUCLEIC ACIDS

Nucleic acids described herein include nucleic acids that are effectiveto modulate salinity and/or oxidative stress tolerance levels whentranscribed in a plant or plant cell. Such nucleic acids include,without limitation, those that encode a salinity and/or oxidative stresstolerance-modulating polypeptide and those that can be used to inhibitexpression of a salinity tolerance and/or oxidative stresstolerance-modulating polypeptide via a nucleic acid based method.

A. Nucleic Acids Encoding Salinity Tolerance and/or Oxidative Stresstolerance-Modulating Polypeptides

Nucleic acids encoding salinity tolerance and/or oxidative stresstolerance-modulating polypeptides are described herein. Such nucleicacids include SEQ ID NOs: 1, 3, 5, 7, 10, 12, 16, 18, 21, 26, 28, 32,34, 40, 46, 48, 51, 53, 55, 57, 59, 61, 65, 67, 70, 72, 75, 77, 79, 82,85, 87, 89, 92, 95, 97, 99, 103, 105, 108, 111, 113, 115, 117, 120, 124,131, 133, 135, 137, 139, 146, 148, 150, 152, 155, 157, 159, 161, and164, as described in more detail below.

A salinity tolerance and/or oxidative stress tolerance-modulatingnucleic acid can comprise the nucleotide sequence set forth in SEQ IDNO: 85. Alternatively, a salinity tolerance and/or oxidative stresstolerance-modulating nucleic acid can be a variant of the nucleic acidhaving the nucleotide sequence set forth in SEQ ID NO: 85. For example,a salinity tolerance and/or oxidative stress tolerance-modulatingnucleic acid can have a nucleotide sequence with at least 80% sequenceidentity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity,to the nucleotide sequence set forth in SEQ ID NO: 85, 87, 89, 92, 95,97, 99, 103, and 105.

A salinity tolerance and/or oxidative stress tolerance-modulatingnucleic acid can comprise the nucleotide sequence set forth in SEQ IDNO: 40. Alternatively, a salinity tolerance and/or oxidative stresstolerance-modulating nucleic acid can be a variant of the nucleic acidhaving the nucleotide sequence set forth in SEQ ID NO: 40. For example,a salinity tolerance and/or oxidative stress tolerance-modulatingnucleic acid can have a nucleotide sequence with at least 80% sequenceidentity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity,to the nucleotide sequence set forth in SEQ ID NO: 40, 46, 48, 51, 53,55, 57, 59, 61, 65, 67, 70, 72, 75, 77, 79, and 82.

A salinity tolerance and/or oxidative stress tolerance-modulatingnucleic acid can comprise the nucleotide sequence set forth in SEQ IDNO: 108. Alternatively, a salinity tolerance and/or oxidative stresstolerance-modulating nucleic acid can be a variant of the nucleic acidhaving the nucleotide sequence set forth in SEQ ID NO: 108. For example,a salinity tolerance and/or oxidative stress tolerance-modulatingnucleic acid can have a nucleotide sequence with at least 80% sequenceidentity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity,to the nucleotide sequence set forth in SEQ ID NO: 108, 111, 113, 115,117, 120, 124, 131, and 133.

A salinity tolerance and/or oxidative stress tolerance-modulatingnucleic acid can comprise the nucleotide sequence set forth in SEQ IDNO: 139. Alternatively, a salinity tolerance and/or oxidative stresstolerance-modulating nucleic acid can be a variant of the nucleic acidhaving the nucleotide sequence set forth in SEQ ID NO: 139. For example,a salinity tolerance and/or oxidative stress tolerance-modulatingnucleic acid can have a nucleotide sequence with at least 80% sequenceidentity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity,to the nucleotide sequence set forth in SEQ ID NO: 135, 137, 139, 146,148, 150, 152, 155, 157, 159, 161, and 164.

A salinity tolerance and/or oxidative stress tolerance-modulatingnucleic acid can comprise the nucleotide sequence set forth in SEQ IDNO: 1. Alternatively, a salinity tolerance and/or oxidative stresstolerance-modulating nucleic acid can be a variant of the nucleic acidhaving the nucleotide sequence set forth in SEQ ID NO: 1. For example, asalinity tolerance and/or oxidative stress tolerance-modulating nucleicacid can have a nucleotide sequence with at least 80% sequence identity,e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to thenucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 10, 12, 16, 18,21, 26, 28, and 32.

A salinity tolerance and/or oxidative stress tolerance-modulatingnucleic acid can comprise the nucleotide sequence set forth in SEQ IDNO: 34. Alternatively, a salinity tolerance and/or oxidative stresstolerance-modulating nucleic acid can be a variant of the nucleic acidhaving the nucleotide sequence set forth in SEQ ID NO: 34. For example,a salinity tolerance and/or oxidative stress tolerance-modulatingnucleic acid can have a nucleotide sequence with at least 80% sequenceidentity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity,to the nucleotide sequence set forth in SEQ ID NO:34.

Isolated nucleic acid molecules can be produced by standard techniques.For example, polymerase chain reaction (PCR) techniques can be used toobtain an isolated nucleic acid containing a nucleotide sequencedescribed herein. PCR can be used to amplify specific sequences from DNAas well as RNA, including sequences from total genomic DNA or totalcellular RNA. Various PCR methods are described, for example, in PCRPrimer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold SpringHarbor Laboratory Press, 1995. Generally, sequence information from theends of the region of interest or beyond is employed to designoligonucleotide primers that are identical or similar in sequence toopposite strands of the template to be amplified. Various PCR strategiesalso are available by which site-specific nucleotide sequencemodifications can be introduced into a template nucleic acid. Isolatednucleic acids also can be chemically synthesized, either as a singlenucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector. Isolated nucleic acids of the invention also canbe obtained by mutagenesis of, e.g., a naturally occurring DNA.

B. Use of Nucleic Acids to Modulate Expression of Polypeptides

-   -   i. Expression of a Salinity Tolerance and/or Oxidative Stress        Tolerance-Modulating Polypeptide

A nucleic acid encoding one of the salinity tolerance and/or oxidativestress tolerance-modulating polypeptides described herein can be used toexpress the polypeptide in a plant species of interest, typically bytransforming a plant cell with a nucleic acid having the coding sequencefor the polypeptide operably linked in sense orientation to one or moreregulatory regions. It will be appreciated that because of thedegeneracy of the genetic code, a number of nucleic acids can encode aparticular salinity tolerance and/or oxidative stresstolerance-modulating polypeptide; i.e., for many amino acids, there ismore than one nucleotide triplet that serves as the codon for the aminoacid. Thus, codons in the coding sequence for a given salinity toleranceand/or oxidative stress tolerance-modulating polypeptide can be modifiedsuch that optimal expression in a particular plant species is obtained,using appropriate codon bias tables for that species.

In some cases, expression of a salinity tolerance and/or oxidativestress tolerance-modulating polypeptide inhibits one or more functionsof an endogenous polypeptide. For example, a nucleic acid that encodes adominant negative polypeptide can be used to inhibit protein function. Adominant negative polypeptide typically is mutated or truncated relativeto an endogenous wild type polypeptide, and its presence in a cellinhibits one or more functions of the wild type polypeptide in thatcell, i.e., the dominant negative polypeptide is genetically dominantand confers a loss of function. The mechanism by which a dominantnegative polypeptide confers such a phenotype can vary but ofteninvolves a protein-protein interaction or a protein-DNA interaction. Forexample, a dominant negative polypeptide can be an enzyme that istruncated relative to a native wild type enzyme, such that the truncatedpolypeptide retains domains involved in binding a first protein butlacks domains involved in binding a second protein. The truncatedpolypeptide is thus unable to properly modulate the activity of thesecond protein. See, e.g., US 2007/0056058. As another example, a pointmutation that results in a non-conservative amino acid substitution in acatalytic domain can result in a dominant negative polypeptide. See,e.g., US 2005/032221. As another example, a dominant negativepolypeptide can be a transcription factor that is truncated relative toa native wild type transcription factor, such that the truncatedpolypeptide retains the DNA binding domain(s) but lacks the activationdomain(s). Such a truncated polypeptide can inhibit the wild typetranscription factor from binding DNA, thereby inhibiting transcriptionactivation.

-   -   ii. Inhibition of Expression of a Salinity Tolerance and/or        Oxidative Stress Tolerance-Modulating Polypeptide

Polynucleotides and recombinant constructs described herein can be usedto inhibit expression of a salinity tolerance and/or oxidative stresstolerance-modulating polypeptide in a plant species of interest. See,e.g., Matzke and Birchler, Nature Reviews Genetics 6:24-35 (2005);Akashi et al., Nature Reviews Mol. Cell Biology 6:413-422 (2005);Mittal, Nature Reviews Genetics 5:355-365 (2004); Dorsett and Tuschl,Nature Reviews Drug Discovery 3: 318-329 (2004); and Nature Reviews RNAinterference collection, October 2005 at nature.com/reviews/focus/mai. Anumber of nucleic acid based methods, including antisense RNA, ribozymedirected RNA cleavage, post-transcriptional gene silencing (PTGS), e.g.,RNA interference (RNAi), and transcriptional gene silencing (TGS) areknown to inhibit gene expression in plants. Antisense technology is onewell-known method. In this method, a nucleic acid segment from a gene tobe repressed is cloned and operably linked to a regulatory region and atranscription termination sequence so that the antisense strand of RNAis transcribed. The recombinant construct is then transformed intoplants, as described herein, and the antisense strand of RNA isproduced. The nucleic acid segment need not be the entire sequence ofthe gene to be repressed, but typically will be substantiallycomplementary to at least a portion of the sense strand of the gene tobe repressed. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Typically, a sequence of at least 30nucleotides is used, e.g., at least 40, 50, 80, 100, 200, 500nucleotides or more.

In another method, a nucleic acid can be transcribed into a ribozyme, orcatalytic RNA, that affects expression of an mRNA. See, U.S. Pat. No.6,423,885. Ribozymes can be designed to specifically pair with virtuallyany target RNA and cleave the phosphodiester backbone at a specificlocation, thereby functionally inactivating the target RNA. Heterologousnucleic acids can encode ribozymes designed to cleave particular mRNAtranscripts, thus preventing expression of a polypeptide. Hammerheadribozymes are useful for destroying particular mRNAs, although variousribozymes that cleave mRNA at site-specific recognition sequences can beused. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target RNA contains a 5′-UG-3′nucleotide sequence. The construction and production of hammerheadribozymes is known in the art. See, for example, U.S. Pat. No. 5,254,678and WO 02/46449 and references cited therein. Hammerhead ribozymesequences can be embedded in a stable RNA such as a transfer RNA (tRNA)to increase cleavage efficiency in vivo. Perriman et al., Proc. Natl.Acad. Sci. USA, 92(13):6175-6179 (1995); de Feyter and Gaudron, Methodsin Molecular Biology, Vol. 74, Chapter 43, “Expressing Ribozymes inPlants”, Edited by Turner, P. C., Humana Press Inc., Totowa, N.J. RNAendoribonucleases which have been described, such as the one that occursnaturally in Tetrahymena thermophila, can be useful. See, for example,U.S. Pat. Nos. 4,987,071 and 6,423,885.

PTGS, e.g., RNAi, can also be used to inhibit the expression of a gene.For example, a construct can be prepared that includes a sequence thatis transcribed into an RNA that can anneal to itself, e.g., a doublestranded RNA having a stem-loop structure. In some embodiments, onestrand of the stem portion of a double stranded RNA comprises a sequencethat is similar or identical to the sense coding sequence of a salinitytolerance and/or oxidative stress tolerance-modulating polypeptide, andthat is from about 10 nucleotides to about 2,500 nucleotides in length.The length of the sequence that is similar or identical to the sensecoding sequence can be from 10 nucleotides to 500 nucleotides, from 15nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides,or from 25 nucleotides to 100 nucleotides. The other strand of the stemportion of a double stranded RNA comprises a sequence that is similar oridentical to the antisense strand of the coding sequence of the salinitytolerance and/or oxidative stress tolerance-modulating polypeptide, andcan have a length that is shorter, the same as, or longer than thecorresponding length of the sense sequence. In some cases, one strand ofthe stem portion of a double stranded RNA comprises a sequence that issimilar or identical to the 3′ or 5′ untranslated region of an mRNAencoding a salinity tolerance and/or oxidative stresstolerance-modulating polypeptide, and the other strand of the stemportion of the double stranded RNA comprises a sequence that is similaror identical to the sequence that is complementary to the 3′ or 5′untranslated region, respectively, of the mRNA encoding the salinitytolerance and/or oxidative stress tolerance-modulating polypeptide. Inother embodiments, one strand of the stem portion of a double strandedRNA comprises a sequence that is similar or identical to the sequence ofan intron in the pre-mRNA encoding a salinity tolerance and/or oxidativestress tolerance-modulating polypeptide, and the other strand of thestem portion comprises a sequence that is similar or identical to thesequence that is complementary to the sequence of the intron in thepre-mRNA. The loop portion of a double stranded RNA can be from 3nucleotides to 5,000 nucleotides, e.g., from 3 nucleotides to 25nucleotides, from 15 nucleotides to 1,000 nucleotides, from 20nucleotides to 500 nucleotides, or from 25 nucleotides to 200nucleotides. The loop portion of the RNA can include an intron. A doublestranded RNA can have zero, one, two, three, four, five, six, seven,eight, nine, ten, or more stem-loop structures. A construct including asequence that is operably linked to a regulatory region and atranscription termination sequence, and that is transcribed into an RNAthat can form a double stranded RNA, is transformed into plants asdescribed herein. Methods for using RNAi to inhibit the expression of agene are known to those of skill in the art. See, e.g., U.S. Pat. Nos.5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588.See also WO 97/01952; WO 98/53083; WO 99/32619; WO 98/36083; and U.S.Patent Publications 20030175965, 20030175783, 20040214330, and20030180945.

Constructs containing regulatory regions operably linked to nucleic acidmolecules in sense orientation can also be used to inhibit theexpression of a gene. The transcription product can be similar oridentical to the sense coding sequence of a salinity tolerance and/oroxidative stress tolerance-modulating polypeptide. The transcriptionproduct can also be unpolyadenylated, lack a 5′ cap structure, orcontain an unsplicable intron. Methods of inhibiting gene expressionusing a full-length cDNA as well as a partial cDNA sequence are known inthe art. See, e.g., U.S. Pat. No. 5,231,020.

In some embodiments, a construct containing a nucleic acid having atleast one strand that is a template for both sense and antisensesequences that are complementary to each other is used to inhibit theexpression of a gene. The sense and antisense sequences can be part of alarger nucleic acid molecule or can be part of separate nucleic acidmolecules having sequences that are not complementary. The sense orantisense sequence can be a sequence that is identical or complementaryto the sequence of an mRNA, the 3′ or 5′ untranslated region of an mRNA,or an intron in a pre-mRNA encoding a salinity tolerance and/oroxidative stress tolerance-modulating polypeptide. In some embodiments,the sense or antisense sequence is identical or complementary to asequence of the regulatory region that drives transcription of the geneencoding a salinity tolerance and/or oxidative stresstolerance-modulating polypeptide. In each case, the sense sequence isthe sequence that is complementary to the antisense sequence.

The sense and antisense sequences can be any length greater than about12 nucleotides (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, or more nucleotides). For example, an antisensesequence can be 21 or 22 nucleotides in length. Typically, the sense andantisense sequences range in length from about 15 nucleotides to about30 nucleotides, e.g., from about 18 nucleotides to about 28 nucleotides,or from about 21 nucleotides to about 25 nucleotides.

In some embodiments, an antisense sequence is a sequence complementaryto an mRNA sequence encoding a salinity tolerance and/or oxidativestress tolerance-modulating polypeptide described herein. The sensesequence complementary to the antisense sequence can be a sequencepresent within the mRNA of the salinity tolerance and/or oxidativestress tolerance-modulating polypeptide. Typically, sense and antisensesequences are designed to correspond to a 15-30 nucleotide sequence of atarget mRNA such that the level of that target mRNA is reduced.

In some embodiments, a construct containing a nucleic acid having atleast one strand that is a template for more than one sense sequence(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sense sequences) can be usedto inhibit the expression of a gene. Likewise, a construct containing anucleic acid having at least one strand that is a template for more thanone antisense sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreantisense sequences) can be used to inhibit the expression of a gene.For example, a construct can contain a nucleic acid having at least onestrand that is a template for two sense sequences and two antisensesequences. The multiple sense sequences can be identical or different,and the multiple antisense sequences can be identical or different. Forexample, a construct can have a nucleic acid having one strand that is atemplate for two identical sense sequences and two identical antisensesequences that are complementary to the two identical sense sequences.Alternatively, an isolated nucleic acid can have one strand that is atemplate for (1) two identical sense sequences 20 nucleotides in length,(2) one antisense sequence that is complementary to the two identicalsense sequences 20 nucleotides in length, (3) a sense sequence 30nucleotides in length, and (4) three identical antisense sequences thatare complementary to the sense sequence 30 nucleotides in length. Theconstructs provided herein can be designed to have any arrangement ofsense and antisense sequences. For example, two identical sensesequences can be followed by two identical antisense sequences or can bepositioned between two identical antisense sequences.

A nucleic acid having at least one strand that is a template for one ormore sense and/or antisense sequences can be operably linked to aregulatory region to drive transcription of an RNA molecule containingthe sense and/or antisense sequence(s). In addition, such a nucleic acidcan be operably linked to a transcription terminator sequence, such asthe terminator of the nopaline synthase (nos) gene. In some cases, tworegulatory regions can direct transcription of two transcripts: one fromthe top strand, and one from the bottom strand. See, for example, Yan etal., Plant Physiol., 141:1508-1518 (2006). The two regulatory regionscan be the same or different. The two transcripts can formdouble-stranded RNA molecules that induce degradation of the target RNA.In some cases, a nucleic acid can be positioned within a T-DNA orplant-derived transfer DNA (P-DNA) such that the left and right T-DNAborder sequences, or the left and right border-like sequences of theP-DNA, flank or are on either side of the nucleic acid. See, US2006/0265788. The nucleic acid sequence between the two regulatoryregions can be from about 15 to about 300 nucleotides in length. In someembodiments, the nucleic acid sequence between the two regulatoryregions is from about 15 to about 200 nucleotides in length, from about15 to about 100 nucleotides in length, from about 15 to about 50nucleotides in length, from about 18 to about 50 nucleotides in length,from about 18 to about 40 nucleotides in length, from about 18 to about30 nucleotides in length, or from about 18 to about 25 nucleotides inlength.

In some nucleic-acid based methods for inhibition of gene expression inplants, a suitable nucleic acid can be a nucleic acid analog. Nucleicacid analogs can be modified at the base moiety, sugar moiety, orphosphate backbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six-membered morpholino ring, or peptidenucleic acids, in which the deoxyphosphate backbone is replaced by apseudopeptide backbone and the four bases are retained. See, forexample, Summerton and Weller, 1997, Antisense Nucleic Acid Drug Dev.,7:187-195; Hyrup et al., Bioorgan. Med. Chem., 4:5-23 (1996). Inaddition, the deoxyphosphate backbone can be replaced with, for example,a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite,or an alkyl phosphotriester backbone.

C. Constructs/Vectors

Recombinant constructs provided herein can be used to transform plantsor plant cells in order to modulate salinity tolerance and/or oxidativestress tolerance levels. A recombinant nucleic acid construct cancomprise a nucleic acid encoding a salinity tolerance and/or oxidativestress tolerance-modulating polypeptide as described herein, operablylinked to a regulatory region suitable for expressing the salinitytolerance and/or oxidative stress tolerance-modulating polypeptide inthe plant or cell. Thus, a nucleic acid can comprise a coding sequencethat encodes any of the salinity tolerance and/or oxidative stresstolerance-modulating polypeptides as set forth in SEQ ID NOs: 1, 3, 5,7, 10, 12, 16, 18, 21, 26, 28, 32, 34, 40, 46, 48, 51, 53, 55, 57, 59,61, 65, 67, 70, 72, 75, 77, 79, 82, 85, 87, 89, 92, 95, 97, 99, 103,105, 108, 111, 113, 115, 117, 120, 124, 131, 133, 135, 137, 139, 146,148, 150, 152, 155, 157, 159, 161, and 164. Examples of nucleic acidsencoding salinity tolerance and/or oxidative stress tolerance-modulatingpolypeptides are set forth in SEQ ID NOs: 2, 4, 6, 8, 9, 11, 13, 14, 15,17, 19, 20, 22, 23, 24, 25, 27, 29, 30, 31, 33, 35, 36, 37, 38, 39, 41,42, 43, 44, 45, 47, 49, 50, 52, 54, 56, 58, 60, 62, 63, 64, 66, 68, 69,71, 73, 74, 76, 78, 80, 81, 83, 84, 86, 88, 90, 91, 93, 94, 96, 98, 100,101, 102, 104, 106, 107, 109, 110, 112, 114, 116, 118, 119, 121, 122,123, 125, 126, 127, 128, 129, 130, 132, 134, 136, 138, 140, 141, 142,143, 144, 145, 147, 149, 151, 153, 154, 156, 158, 160, 162, 163, 165,166, 167, 168, and amino acid coordinates 1 to 135 of SEQ ID NO: 140.The salinity tolerance and/or oxidative stress tolerance-modulatingpolypeptide encoded by a recombinant nucleic acid can be a nativesalinity tolerance and/or oxidative stress tolerance-modulatingpolypeptide, or can be heterologous to the cell. In some cases, therecombinant construct contains a nucleic acid that inhibits expressionof a salinity tolerance and/or oxidative stress tolerance-modulatingpolypeptide, operably linked to a regulatory region. Examples ofsuitable regulatory regions are described in the section entitled“Regulatory Regions.”

Vectors containing recombinant nucleic acid constructs such as thosedescribed herein also are provided. Suitable vector backbones include,for example, those routinely used in the art such as plasmids, viruses,artificial chromosomes, BACs, YACs, or PACs. Suitable expression vectorsinclude, without limitation, plasmids and viral vectors derived from,for example, bacteriophage, baculoviruses, and retroviruses. Numerousvectors and expression systems are commercially available from suchcorporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.),Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies(Carlsbad, Calif.).

The vectors provided herein also can include, for example, origins ofreplication, scaffold attachment regions (SARs), and/or markers. Amarker gene can confer a selectable phenotype on a plant cell. Forexample, a marker can confer biocide resistance, such as resistance toan antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin), or anherbicide (e.g., glyphosate, chlorsulfuron or phosphinothricin). Inaddition, an expression vector can include a tag sequence designed tofacilitate manipulation or detection (e.g., purification orlocalization) of the expressed polypeptide. Tag sequences, such asluciferase, β-glucuronidase (GUS), green fluorescent protein (GFP),glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, orFlag™ tag (Kodak, New Haven, Conn.) sequences typically are expressed asa fusion with the encoded polypeptide. Such tags can be insertedanywhere within the polypeptide, including at either the carboxyl oramino terminus.

D. Regulatory Regions

The choice of regulatory regions to be included in a recombinantconstruct depends upon several factors, including, but not limited to,efficiency, selectability, inducibility, desired expression level, andcell- or tissue-preferential expression. It is a routine matter for oneof skill in the art to modulate the expression of a coding sequence byappropriately selecting and positioning regulatory regions relative tothe coding sequence. Transcription of a nucleic acid can be modulated ina similar manner.

Some suitable promoters initiate transcription only, or predominantly,in certain cell types. The choice of regulatory regions to be includedin a recombinant construct depends upon several factors, including, butnot limited to, efficiency, selectability, inducibility, desiredexpression level, and cell- or tissue-preferential expression. It is aroutine matter for one of skill in the art to modulate the expression ofa coding sequence by appropriately selecting and positioning regulatoryregions relative to the coding sequence. Transcription of a nucleic acidcan be modulated in a similar manner.

Some suitable regulatory regions initiate transcription only, orpredominantly, in certain cell types. Methods for identifying andcharacterizing regulatory regions in plant genomic DNA are known,including, for example, those described in the following references:Jordano et al., Plant Cell, 1:855-866 (1989); Bustos et al., Plant Cell,1:839-854 (1989); Green et al., EMBO J., 7:4035-4044 (1988); Meier etal., Plant Cell, 3:309-316 (1991); and Zhang et al., Plant Physiology,110:1069-1079 (1996).

Examples of various classes of regulatory regions are described below.Some of the regulatory regions indicated below as well as additionalregulatory regions are described in more detail in U.S. patentapplication Ser. Nos. 60/505,689; 60/518,075; 60/544,771; 60/558,869;60/583,691; 60/619,181; 60/637,140; 60/757,544; 60/776,307; 10/957,569;11/058,689; 11/172,703; 11/208,308; 11/274,890; 60/583,609; 60/612,891;11/097,589; 11/233,726; 11/408,791; 11/414,142; 10/950,321; 11/360,017;PCT/US05/011105; PCT/US05/23639; PCT/US05/034308; PCT/US05/034343; andPCT/US06/038236; PCT/US06/040572; and PCT/US07/62762.

For example, the sequences of regulatory regions p326, YP0144, YP0190,p13879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848, PT0633,YP0128, YP0275, PT0660, PT0683, PT0758, PT0613, PT0672, PT0688, PT0837,YP0092, PT0676, PT0708, YP0396, YP0007, YP0111, YP0103, YP0028, YP0121,YP0008, YP0039, YP0115, YP0119, YP0120, YP0374, YP0101, YP0102, YP0110,YP0117, YP0137, YP0285, YP0212, YP0097, YP0107, YP0088, YP0143, YP0156,PT0650, PT0695, PT0723, PT0838, PT0879, PT0740, PT0535, PT0668, PT0886,PT0585, YP0381, YP0337, PT0710, YP0356, YP0385, YP0384, YP0286, YP0377,PD1367, PT0863, PT0829, PT0665, PT0678, YP0086, YP0188, YP0263, PT0743and YP0096 are set forth in the sequence listing of PCT/US06/040572; thesequence of regulatory region PT0625 is set forth in the sequencelisting of PCT/US05/034343; the sequences of regulatory regions PT0623,YP0388, YP0087, YP0093, YP0108, YP0022 and YP0080 are set forth in thesequence listing of U.S. patent application Ser. No. 11/172,703; thesequence of regulatory region PR0924 is set forth in the sequencelisting of PCT/US07/62762; and the sequences of regulatory regionsp530c10, pOsFIE2-2, pOsMEA, pOsYp102, and pOsYp285 are set forth in thesequence listing of PCT/US06/038236.

It will be appreciated that a regulatory region may meet criteria forone classification based on its activity in one plant species, and yetmeet criteria for a different classification based on its activity inanother plant species.

-   -   i. Broadly Expressing Promoters

A promoter can be said to be “broadly expressing” when it promotestranscription in many, but not necessarily all, plant tissues. Forexample, a broadly expressing promoter can promote transcription of anoperably linked sequence in one or more of the shoot, shoot tip (apex),and leaves, but weakly or not at all in tissues such as roots or stems.As another example, a broadly expressing promoter can promotetranscription of an operably linked sequence in one or more of the stem,shoot, shoot tip (apex), and leaves, but can promote transcriptionweakly or not at all in tissues such as reproductive tissues of flowersand developing seeds. Non-limiting examples of broadly expressingpromoters that can be included in the nucleic acid constructs providedherein include the p326, YP0144, YP0190, p13879, YP0050, p32449, 21876,YP0158, YP0214, YP0380, PT0848, and PT0633 promoters. Additionalexamples include the cauliflower mosaic virus (CaMV) 35S promoter, themannopine synthase (MAS) promoter, the 1′ or 2′ promoters derived fromT-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34Spromoter, actin promoters such as the rice actin promoter, and ubiquitinpromoters such as the maize ubiquitin-1 promoter. In some cases, theCaMV 35S promoter is excluded from the category of broadly expressingpromoters.

-   -   ii. Root Promoters

Root-active promoters confer transcription in root tissue, e.g., rootendodermis, root epidermis, or root vascular tissues. In someembodiments, root-active promoters are root-preferential promoters,i.e., confer transcription only or predominantly in root tissue.Root-preferential promoters include the YP0128, YP0275, PT0625, PT0660,PT0683, and PT0758 promoters. Other root-preferential promoters includethe PT0613, PT0672, PT0688, and PT0837 promoters, which drivetranscription primarily in root tissue and to a lesser extent in ovulesand/or seeds. Other examples of root-preferential promoters include theroot-specific subdomains of the CaMV 35S promoter (Lam et al., Proc.Natl. Acad. Sci. USA, 86:7890-7894 (1989)), root cell specific promotersreported by Conkling et al., Plant Physiol., 93:1203-1211 (1990), andthe tobacco RD2 promoter.

-   -   iii. Maturing Endosperm Promoters

In some embodiments, promoters that drive transcription in maturingendosperm can be useful. Transcription from a maturing endospermpromoter typically begins after fertilization and occurs primarily inendosperm tissue during seed development and is typically highest duringthe cellularization phase. Most suitable are promoters that are activepredominantly in maturing endosperm, although promoters that are alsoactive in other tissues can sometimes be used. Non-limiting examples ofmaturing endosperm promoters that can be included in the nucleic acidconstructs provided herein include the napin promoter, the Arcelin-5promoter, the phaseolin promoter (Bustos et al., Plant Cell,1(9):839-853 (1989)), the soybean trypsin inhibitor promoter (Riggs etal., Plant Cell, 1(6):609-621 (1989)), the ACP promoter (Baerson et al.,Plant Mol. Biol., 22(2):255-267 (1993)), the stearoyl-ACP desaturasepromoter (Slocombe et al., Plant Physiol., 104(4):167-176 (1994)), thesoybean α′ subunit of β-conglycinin promoter (Chen et al., Proc. Natl.Acad. Sci. USA, 83:8560-8564 (1986)), the oleosin promoter (Hong et al.,Plant Mol. Biol., 34(3):549-555 (1997)), and zein promoters, such as the15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kDzein promoter and 27 kD zein promoter. Also suitable are the Osgt-1promoter from the rice glutelin-1 gene (Zheng et al., Mol. Cell Biol.,13:5829-5842 (1993)), the beta-amylase promoter, and the barley hordeinpromoter. Other maturing endosperm promoters include the YP0092, PT0676,and PT0708 promoters.

-   -   iv. Ovary Tissue Promoters

Promoters that are active in ovary tissues such as the ovule wall andmesocarp can also be useful, e.g., a polygalacturonidase promoter, thebanana TRX promoter, the melon actin promoter, YP0396, and PT0623.Examples of promoters that are active primarily in ovules includeYP0007, YP0111, YP0092, YP0103, YP0028, YP0121, YP0008, YP0039, YP0115,YP0119, YP0120, and YP0374.

-   -   v. Embryo Sac/Early Endosperm Promoters

To achieve expression in embryo sac/early endosperm, regulatory regionscan be used that are active in polar nuclei and/or the central cell, orin precursors to polar nuclei, but not in egg cells or precursors to eggcells. Most suitable are promoters that drive expression only orpredominantly in polar nuclei or precursors thereto and/or the centralcell. A pattern of transcription that extends from polar nuclei intoearly endosperm development can also be found with embryo sac/earlyendosperm-preferential promoters, although transcription typicallydecreases significantly in later endosperm development during and afterthe cellularization phase. Expression in the zygote or developing embryotypically is not present with embryo sac/early endosperm promoters.

Promoters that may be suitable include those derived from the followinggenes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsisatmycl (see, Urao (1996) Plant Mol. Biol., 32:571-57; Conceicao (1994)Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); ArabidopsisMEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Pat. No.6,906,244). Other promoters that may be suitable include those derivedfrom the following genes: maize MAC1 (see, Sheridan (1996) Genetics,142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) PlantMol. Biol., 22:10131-1038). Other promoters include the followingArabidopsis promoters: YP0039, YP0101, YP0102, YP0110, YP0117, YP0119,YP0137, DME, YP0285, and YP0212. Other promoters that may be usefulinclude the following rice promoters: p530c10, pOsFIE2-2, pOsMEA,pOsYp102, and pOsYp285.

-   -   vi. Embryo Promoters

Regulatory regions that preferentially drive transcription in zygoticcells following fertilization can provide embryo-preferentialexpression. Most suitable are promoters that preferentially drivetranscription in early stage embryos prior to the heart stage, butexpression in late stage and maturing embryos is also suitable.Embryo-preferential promoters include the barley lipid transfer protein(Ltp1) promoter (Plant Cell Rep (2001) 20:647-654), YP0097, YP0107,YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838, PT0879, andPT0740.

-   -   vii. Photosynthetic Tissue Promoters

Promoters active in photosynthetic tissue confer transcription in greentissues such as leaves and stems. Most suitable are promoters that driveexpression only or predominantly in such tissues. Examples of suchpromoters include the ribulose-1,5-bisphosphate carboxylase (RbcS)promoters such as the RbcS promoter from eastern larch (Larix laricina),the pine cab6 promoter (Yamamoto et al., Plant Cell Physiol., 35:773-778(1994)), the Cab-1 promoter from wheat (Fejes et al., Plant Mol. Biol.,15:921-932 (1990)), the CAB-1 promoter from spinach (Lubberstedt et al.,Plant Physiol., 104:997-1006 (1994)), the cab1R promoter from rice (Luanet al., Plant Cell, 4:971-981 (1992)), the pyruvate orthophosphatedikinase (PPDK) promoter from corn (Matsuoka et al., Proc. Natl. Acad.Sci. USA, 90:9586-9590 (1993)), the tobacco Lhcb1*2 promoter (Cerdan etal., Plant Mol. Biol., 33:245-255 (1997)), the Arabidopsis thaliana SUC2sucrose-H+ symporter promoter (Truernit et al., Planta, 196:564-570(1995)), and thylakoid membrane protein promoters from spinach (psaD,psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS). Other photosynthetic tissuepromoters include PT0535, PT0668, PT0886, YP0144, YP0380 and PT0585.

-   -   viii. Vascular Tissue Promoters

Examples of promoters that have high or preferential activity invascular bundles include YP0087, YP0093, YP0108, YP0022, and YP0080.Other vascular tissue-preferential promoters include the glycine-richcell wall protein GRP 1.8 promoter (Keller and Baumgartner, Plant Cell,3(10):1051-1061 (1991)), the Commelina yellow mottle virus (CoYMV)promoter (Medberry et al., Plant Cell, 4(2):185-192 (1992)), and therice tungro bacilliform virus (RTBV) promoter (Dai et al., Proc. Natl.Acad. Sci. USA, 101(2):687-692 (2004)).

-   -   ix. Inducible Promoters

Inducible promoters confer transcription in response to external stimulisuch as chemical agents or environmental stimuli. For example, induciblepromoters can confer transcription in response to hormones such asgiberellic acid or ethylene, or in response to light or drought.Examples of drought-inducible promoters include YP0380, PT0848, YP0381,YP0337, PT0633, YP0374, PT0710, YP0356, YP0385, YP0396, YP0388, YP0384,PT0688, YP0286, YP0377, PD1367, and PD0901. Examples ofnitrogen-inducible promoters include PT0863, PT0829, PT0665, and PT0886.Examples of shade-inducible promoters include PR0924 and PT0678. Anexample of a promoter induced by salt is rd29A (Kasuga et al. (1999)Nature Biotech 17: 287-291).

-   -   x. Basal Promoters

A basal promoter is the minimal sequence necessary for assembly of atranscription complex required for transcription initiation. Basalpromoters frequently include a “TATA box” element that may be locatedbetween about 15 and about 35 nucleotides upstream from the site oftranscription initiation. Basal promoters also may include a “CCAAT box”element (typically the sequence CCAAT) and/or a GGGCG sequence, whichcan be located between about 40 and about 200 nucleotides, typicallyabout 60 to about 120 nucleotides, upstream from the transcription startsite.

-   -   xi. Other Promoters

Other classes of promoters include, but are not limited to,shoot-preferential, callus-preferential, trichome cell-preferential,guard cell-preferential such as PT0678, tuber-preferential, parenchymacell-preferential, and senescence-preferential promoters. Promotersdesignated YP0086, YP0188, YP0263, PT0758, PT0743, PT0829, YP0119, andYP0096, as described in the above-referenced patent applications, mayalso be useful.

-   -   xii. Other Regulatory Regions

A 5′ untranslated region (UTR) can be included in nucleic acidconstructs described herein. A 5′ UTR is transcribed, but is nottranslated, and lies between the start site of the transcript and thetranslation initiation codon and may include the +1 nucleotide. A 3′ UTRcan be positioned between the translation termination codon and the endof the transcript. UTRs can have particular functions such as increasingmRNA stability or attenuating translation. Examples of 3′ UTRs include,but are not limited to, polyadenylation signals and transcriptiontermination sequences, e.g., a nopaline synthase termination sequence.

It will be understood that more than one regulatory region may bepresent in a recombinant polynucleotide, e.g., introns, enhancers,upstream activation regions, transcription terminators, and inducibleelements. Thus, for example, more than one regulatory region can beoperably linked to the sequence of a polynucleotide encoding a saltand/or oxidative stress tolerance modulating polypeptide.

Regulatory regions, such as promoters for endogenous genes, can beobtained by chemical synthesis or by subcloning from a genomic DNA thatincludes such a regulatory region. A nucleic acid comprising such aregulatory region can also include flanking sequences that containrestriction enzyme sites that facilitate subsequent manipulation.

Alternatively, misexpression can be accomplished using a two componentsystem, whereby the first component consists of a transgenic plantcomprising a transcriptional activator operatively linked to a promoterand the second component consists of a transgenic plant that comprise anucleic acid molecule of the invention operatively linked to thetarget-binding sequence/region of the transcriptional activator. The twotransgenic plants are crossed and the nucleic acid molecule of theinvention is expressed in the progeny of the plant. In anotheralternative embodiment of the present invention, the misexpression canbe accomplished by having the sequences of the two component systemtransformed in one transgenic plant line.

IV. TRANSGENIC PLANTS AND PLANT CELLS

A. Transformation

The invention also features transgenic plant cells and plants comprisingat least one recombinant nucleic acid construct described herein. Aplant or plant cell can be transformed by having a construct integratedinto its genome, i.e., can be stably transformed. Stably transformedcells typically retain the introduced nucleic acid with each celldivision. A plant or plant cell can also be transiently transformed suchthat the construct is not integrated into its genome. Transientlytransformed cells typically lose all or some portion of the introducednucleic acid construct with each cell division such that the introducednucleic acid cannot be detected in daughter cells after a sufficientnumber of cell divisions. Both transiently transformed and stablytransformed transgenic plants and plant cells can be useful in themethods described herein.

Transgenic plant cells used in methods described herein can constitutepart or all of a whole plant. Such plants can be grown in a mannersuitable for the species under consideration, either in a growthchamber, a greenhouse, or in a field. Transgenic plants can be bred asdesired for a particular purpose, e.g., to introduce a recombinantnucleic acid into other lines, to transfer a recombinant nucleic acid toother species, or for further selection of other desirable traits.Alternatively, transgenic plants can be propagated vegetatively forthose species amenable to such techniques. As used herein, a transgenicplant also refers to progeny of an initial transgenic plant having thetransgene. Seeds produced by a transgenic plant can be grown and thenselfed (or outcrossed and selfed) to obtain seeds homozygous for thenucleic acid construct.

Transgenic plants can be grown in suspension culture, or tissue or organculture. For the purposes of this invention, solid and/or liquid tissueculture techniques can be used. When using solid medium, transgenicplant cells can be placed directly onto the medium or can be placed ontoa filter that is then placed in contact with the medium. When usingliquid medium, transgenic plant cells can be placed onto a flotationdevice, e.g., a porous membrane that contacts the liquid medium. A solidmedium can be, for example, Murashige and Skoog (MS) medium containingagar and a suitable concentration of an auxin, e.g.,2,4-dichlorophenoxyacetic acid (2,4-D), and a suitable concentration ofa cytokinin, e.g., kinetin.

When transiently transformed plant cells are used, a reporter sequenceencoding a reporter polypeptide having a reporter activity can beincluded in the transformation procedure and an assay for reporteractivity or expression can be performed at a suitable time aftertransformation. A suitable time for conducting the assay typically isabout 1-21 days after transformation, e.g., about 1-14 days, about 1-7days, or about 1-3 days. The use of transient assays is particularlyconvenient for rapid analysis in different species, or to confirmexpression of a heterologous salinity tolerance and/or oxidative stresstolerance-modulating polypeptide whose expression has not previouslybeen confirmed in particular recipient cells.

Techniques for introducing nucleic acids into monocotyledonous anddicotyledonous plants are known in the art, and include, withoutlimitation, Agrobacterium-mediated transformation, viral vector-mediatedtransformation, electroporation and particle gun transformation, e.g.,U.S. Pat. Nos. 5,538,880; 5,204,253; 6,329,571 and 6,013,863. If a cellor cultured tissue is used as the recipient tissue for transformation,plants can be regenerated from transformed cultures if desired, bytechniques known to those skilled in the art.

B. Screening/Selection

A population of transgenic plants can be screened and/or selected forthose members of the population that have a trait or phenotype conferredby expression of the transgene. For example, a population of progeny ofa single transformation event can be screened for those plants having adesired level of expression of a salinity tolerance and/or oxidativestress tolerance-modulating polypeptide or nucleic acid. Physical andbiochemical methods can be used to identify expression levels. Theseinclude Southern analysis or PCR amplification for detection of apolynucleotide; Northern blots, S1 RNase protection, primer-extension,or RT-PCR amplification for detecting RNA transcripts; enzymatic assaysfor detecting enzyme or ribozyme activity of polypeptides andpolynucleotides; and protein gel electrophoresis, Western blots,immunoprecipitation, and enzyme-linked immunoassays to detectpolypeptides. Other techniques such as in situ hybridization, enzymestaining, and immunostaining also can be used to detect the presence orexpression of polypeptides and/or polynucleotides. Methods forperforming all of the referenced techniques are known. As analternative, a population of plants comprising independenttransformation events can be screened for those plants having a desiredtrait, such as a modulated level of salinity tolerance and/or oxidativestress tolerance. Selection and/or screening can be carried out over oneor more generations, and/or in more than one geographic location. Insome cases, transgenic plants can be grown and selected under conditionswhich induce a desired phenotype or are otherwise necessary to produce adesired phenotype in a transgenic plant. In addition, selection and/orscreening can be applied during a particular developmental stage inwhich the phenotype is expected to be exhibited by the plant. Selectionand/or screening can be carried out to choose those transgenic plantshaving a statistically significant difference in a salinity toleranceand/or oxidative stress tolerance level relative to a control plant thatlacks the transgene. Selected or screened transgenic plants have analtered phenotype as compared to a corresponding control plant, asdescribed in the “Transgenic Plant Phenotypes” section herein.

A population of transgenic plants can be screened and/or selected forthose members of the population that have a trait or phenotype conferredby expression of the transgene. For example, a population of progeny ofa single transformation event can be screened for those plants having adesired level of expression of a saline and/or oxidative stresstolerance-modulating polypeptide and/or nucleic acid. Physical andbiochemical methods can be used to identify expression levels. Theseinclude Southern analysis or PCR amplification for detection of apolynucleotide; Northern blots, S1 RNase protection, primer-extension,or RT-PCR amplification for detecting RNA transcripts; enzymatic assaysfor detecting enzyme or ribozyme activity of polypeptides andpolynucleotides; and protein gel electrophoresis, Western blots,immunoprecipitation, and enzyme-linked immunoassays to detectpolypeptides. Other techniques such as in situ hybridization, enzymestaining, and immunostaining also can be used to detect the presence orexpression of polypeptides and/or polynucleotides. Methods forperforming all of the referenced techniques are known. As analternative, a population of plants comprising independenttransformation events can be screened for those plants having a desiredtrait, such as a modulated level of saline and/or oxidative stresstolerance. Selection and/or screening can be carried out over one ormore generations, and/or in more than one geographic location. In somecases, transgenic plants can be grown and selected under conditionswhich induce a desired phenotype or are otherwise necessary to produce adesired phenotype in a transgenic plant. In addition, selection and/orscreening can be applied during a particular developmental stage inwhich the phenotype is expected to be exhibited by the plant. Selectionand/or screening can be carried out to choose those transgenic plantshaving a statistically significant difference in a saline and/oroxidative stress tolerance level relative to a control plant that lacksthe transgene. Selected or screened transgenic plants have an alteredphenotype as compared to a corresponding control plant, as described inthe “Transgenic Plant Phenotypes” section herein.

C. Plant Species

The polynucleotides and vectors described herein can be used totransform a number of monocotyledonous and dicotyledonous plants andplant cell systems, including species from one of the followingfamilies: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae,Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae,Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae,Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae,Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae,Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae,Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae,Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae,Theaceae, or Vitaceae.

Suitable species may include members of the genus Abelmoschus, Abies,Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon,Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula,Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus,Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum,Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis,Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus,Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea,Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus,Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa,Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia,Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus,Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum,Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale,Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

Suitable species include Panicum spp., Sorghum spp., Miscanthus spp.,Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii (bigbluestem), Pennisetum purpureum (elephant grass), Phalaris arundinacea(reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea(tall fescue), Spartina pectinata (prairie cord-grass), Medicago sativa(alfalfa), Arundo donax (giant reed), Secale cereale (rye), Salix spp.(willow), Eucalyptus spp. (eucalyptus), Triticosecale (triticum—wheat Xrye) and bamboo.

Suitable species also include Helianthus annuus (sunflower), Carthamustinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis(castor), Elaeis guineensis (palm), Linum usitatissimum (flax), andBrassica juncea.

Suitable species also include Beta vulgaris (sugarbeet), and Manihotesculenta (cassava).

Suitable species also include Lycopersicon esculentum (tomato), Lactucasativa (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato),Brassica oleracea (broccoli, cauliflower, brusselsprouts), Camelliasinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa),Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus(pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion),Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), andSolanum melongena (eggplant).

Suitable species also include Papaver somniferum (opium poppy), Papaverorientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabissativa, Camptotheca acuminate, Catharanthus roseus, Vinca rosea,Cinchona officinalis, Colchicum autumnale, Veratrum californica.,Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographispaniculata, Atropa belladonna, Datura stomonium, Berberis spp.,Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca,Galanthus wornorii, Scopolia spp., Lycopodium serratum (=Huperziaserrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp.,Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis,Chrysanthemum parthenium, Coleus forskohlii, and Tanacetum parthenium.

Suitable species also include Parthenium argentatum (guayule), Heveaspp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixaorellana, and Alstroemeria spp.

Suitable species also include Rosa spp. (rose), Dianthus caryophyllus(carnation), Petunia spp. (petunia) and Poinsettia pulcherrima(poinsettia).

Suitable species also include Nicotiana tabacum (tobacco), Lupinus albus(lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populustremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp.(maple, Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp.(ryegrass) and Phleum pratense (timothy).

Thus, the methods and compositions can be used over a broad range ofplant species, including species from the dicot genera Brassica,Carthamus, Glycine, Gossypium, Helianthus, Jatropha, Parthenium,Populus, and Ricinus; and the monocot genera Elaeis, Festuca, Hordeum,Lolium, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum, Secale,Sorghum, Triticosecale, Triticum, and Zea. In some embodiments, a plantis a member of the species Panicum virgatum (switchgrass), Sorghumbicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus),Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays(corn), Glycine max (soybean), Brassica napus (canola), Triticumaestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice),Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris(sugarbeet), or Pennisetum glaucum (pearl millet).

In certain embodiments, the polynucleotides and vectors described hereincan be used to transform a number of monocotyledonous and dicotyledonousplants and plant cell systems, wherein such plants are hybrids ofdifferent species or varieties of a species (e.g., Saccharum sp. XMiscanthus sp.)

D. Transgenic Plant Phenotypes

In some embodiments, a plant in which expression of a salinity and/oroxidative stress modulating polypeptide is modulated can have increasedlevels of tolerance to salinity and/or oxidative stress. For example, asalinity and/or oxidative stress-modulating polypeptide described hereincan be expressed in a transgenic plant, resulting in increased levels oftolerance to salinity and/or oxidative stress. The salinity and/oroxidative stress tolerance levels can be increased by at least 2percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 percent, ascompared to those levels in a corresponding control plant that does notexpress the transgene.

The nucleic acid molecules and polypeptides of the present invention areof interest because when the nucleic acid molecules are mis-expressed(i.e., when expressed at a non-natural location or in an increased ordecreased amount relative to wild-type) they produce plants that exhibitimproved salt tolerance and/or oxidation tolerance as compared towild-type plants, as evidenced in part by the results of variousexperiments disclosed below. In particular, plants transformed with thenucleic acid molecules and polypeptides of the present invention canhave any of a number of modified characteristics as compared towild-type plants. Examples of modified characteristics includephotosynthetic efficiency, seedling area, and biomass as it may bemeasured by plant height, leaf or rosette area, or dry mass. Themodified characteristics may be observed and measured at different plantdevelopmental stages, e.g. seed, seedling, bolting, senescense, etc.Often, salt or oxidative tolerance can be expressed as ratios orcombinations of measurements, such as salt growth index values, orsalicylic acid growth index values. For example, plants transformed withthe sequences of the present invention can exhibit increases in SGI,seedling area and/or SAGI values of at least 5%, at least 10%, at least25%, at least 50%, at least 75%, at least 100%, at least 200%, at least300%, at least 400%, or even at least 500%. These traits can be used toexploit or maximize plant products. For example, the nucleic acidmolecules and polypeptides of the present invention are used to increasethe expression of genes that cause the plant to have improved biomass,growth rate and/or seedling vigor in saline and/or oxidative conditions,in comparison to wild type plants under the same conditions.

Because the disclosed sequences and methods increase vegetative growthand growth rate in saline and/or oxidative conditions, the disclosedmethods can be used to enhance plant growth in plants grown in salineand/or oxidative conditions. For example, plants of the presentinvention show, under saline and/or oxidative conditions, increasedphotosynthetic efficiency and increased seedling area as compared to aplant of the same species that is not genetically modified forsubstantial vegetative growth. Examples of increases in biomassproduction include increases of at least 5%, at least 20%, or even atleast 50%, when compared to an amount of biomass production by awild-type plant of the same species under identical conditions.

Typically, a difference in the amount of tolerance to salinity and/oroxidative stress in a transgenic plant or cell relative to a controlplant or cell is considered statistically significant at p≤0.05 with anappropriate parametric or non-parametric statistic, e.g., Chi-squaretest, Student's t-test, Mann-Whitney test, or F-test. In someembodiments, a difference in the amount of tolerance to salinity and/oroxidative stress is statistically significant at p<0.01, p<0.005, orp<0.001.

The phenotype of a transgenic plant is evaluated relative to a controlplant. A plant is said “not to express” a polypeptide when the plantexhibits less than 10%, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, 0.1%, 0.01%, or 0.001%, of the amount of polypeptide or mRNAencoding the polypeptide exhibited by the plant of interest. Expressioncan be evaluated using methods including, for example, RT-PCR, Northernblots, S1 RNase protection, primer extensions, Western blots, proteingel electrophoresis, immunoprecipitation, enzyme-linked immunoassays,chip assays, and mass spectrometry. It should be noted that if apolypeptide is expressed under the control of a tissue-preferential orbroadly expressing promoter, expression can be evaluated in the entireplant or in a selected tissue. Similarly, if a polypeptide is expressedat a particular time, e.g., at a particular time in development or uponinduction, expression can be evaluated selectively at a desired timeperiod.

V. PLANT BREEDING

Genetic polymorphisms are discrete allelic sequence differences in apopulation. Typically, an allele that is present at 1% or greater isconsidered to be a genetic polymorphism. The discovery that polypeptidesdisclosed herein can modulate salinity tolerance and/or oxidative stresstolerance content is useful in plant breeding, because geneticpolymorphisms exhibiting a degree of linkage with loci for suchpolypeptides are more likely to be correlated with variation in asalinity tolerance and/or oxidative stress tolerance trait. For example,genetic polymorphisms linked to the loci for such polypeptides are morelikely to be useful in marker-assisted breeding programs to create lineshaving a desired modulation in the salinity tolerance and/or oxidativestress tolerance traits.

Thus, one aspect of the invention includes methods of identifyingwhether one or more genetic polymorphisms are associated with variationin a salinity tolerance and/or oxidative stress tolerance trait. Suchmethods involve determining whether genetic polymorphisms in a givenpopulation exhibit linkage with the locus for one of the polypeptidesdepicted in FIGS. 1 thru 6 and/or a functional homolog thereof, such as,but not limited to, those in the Sequence Listing. The correlation ismeasured between variation in the salinity tolerance and/or oxidativestress tolerance traits in plants of the population and the presence ofthe genetic polymorphism(s) in plants of the population, therebyidentifying whether or not the genetic polymorphism(s) are associatedwith variation for the traits. If the presence of a particular allele isstatistically significantly correlated with a desired modulation in thesalinity tolerance and/or oxidative stress tolerance traits, the alleleis associated with variation for one or both of the traits and is usefulas a marker for one or more of the traits. If, on the other hand, thepresence of a particular allele is not significantly correlated with thedesired modulation, the allele is not associated with variation for oneor more of the traits and is not useful as a marker.

Such methods are applicable to populations containing the naturallyoccurring endogenous polypeptide rather than an exogenous nucleic acidencoding the polypeptide, i.e., populations that are not transgenic forthe exogenous nucleic acid. It will be appreciated, however, thatpopulations suitable for use in the methods may contain a transgene foranother, different trait, e.g., herbicide resistance.

Genetic polymorphisms that are useful in such methods include simplesequence repeats (SSRs, or microsatellites), rapid amplification ofpolymorphic DNA (RAPDs), single nucleotide polymorphisms (SNPs),amplified fragment length polymorphisms (AFLPs) and restriction fragmentlength polymorphisms (RFLPs). SSR polymorphisms can be identified, forexample, by making sequence specific probes and amplifying template DNAfrom individuals in the population of interest by PCR. If the probesflank an SSR in the population, PCR products of different sizes will beproduced. See, e.g., U.S. Pat. No. 5,766,847. Alternatively, SSRpolymorphisms can be identified by using PCR product(s) as a probeagainst Southern blots from different individuals in the population.See, U. H. Refseth et al., (1997) Electrophoresis 18: 1519. Theidentification of RFLPs is discussed, for example, in Alonso-Blanco etal. (Methods in Molecular Biology, vol. 82, “Arabidopsis Protocols”, pp.137-146, J. M. Martinez-Zapater and J. Salinas, eds., c. 1998 by HumanaPress, Totowa, N.J.); Burr (“Mapping Genes with Recombinant Inbreds”,pp. 249-254, in Freeling, M. and V. Walbot (Ed.), The Maize Handbook, c.1994 by Springer-Verlag New York, Inc.: New York, N.Y., USA; BerlinGermany; Burr et al. Genetics (1998) 118: 519; and Gardiner, J. et al.,(1993) Genetics 134: 917). The identification of AFLPs is discussed, forexample, in EP 0 534 858 and U.S. Pat. No. 5,878,215.

In some embodiments, the methods are directed to breeding a plant line.Such methods use genetic polymorphisms identified as described above ina marker assisted breeding program to facilitate the development oflines that have a desired alteration in the salinity tolerance and/oroxidative stress tolerance trait(s). Once a suitable geneticpolymorphism is identified as being associated with variation for thetrait, one or more individual plants are identified that possess thepolymorphic allele correlated with the desired variation. Those plantsare then used in a breeding program to combine the polymorphic allelewith a plurality of other alleles at other loci that are correlated withthe desired variation. Techniques suitable for use in a plant breedingprogram are known in the art and include, without limitation,backcrossing, mass selection, pedigree breeding, bulk selection,crossing to another population and recurrent selection. These techniquescan be used alone or in combination with one or more other techniques ina breeding program. Thus, each identified plants is selfed or crossed adifferent plant to produce seed which is then germinated to form progenyplants. At least one such progeny plant is then selfed or crossed with adifferent plant to form a subsequent progeny generation. The breedingprogram can repeat the steps of selfing or outcrossing for an additional0 to 5 generations as appropriate in order to achieve the desireduniformity and stability in the resulting plant line, which retains thepolymorphic allele. In most breeding programs, analysis for theparticular polymorphic allele will be carried out in each generation,although analysis can be carried out in alternate generations ifdesired.

In some cases, selection for other useful traits is also carried out,e.g., selection for fungal resistance or bacterial resistance. Selectionfor such other traits can be carried out before, during or afteridentification of individual plants that possess the desired polymorphicallele.

VI. ARTICLES OF MANUFACTURE

Transgenic plants provided herein have various uses in the agriculturaland energy production industries. For example, transgenic plantsdescribed herein can be used to make animal feed and food products. Suchplants, however, are often particularly useful as a feedstock for energyproduction.

Transgenic plants described herein often produce higher yields of grainand/or biomass per hectare, relative to control plants that lack theexogenous nucleic acid. In some embodiments, such transgenic plantsprovide equivalent or even increased yields of grain and/or biomass perhectare relative to control plants when grown under conditions ofreduced inputs such as fertilizer and/or water. Thus, such transgenicplants can be used to provide yield stability at a lower input costand/or under environmentally stressful conditions such as drought. Insome embodiments, plants described herein have a composition thatpermits more efficient processing into free sugars, and subsequentlyethanol, for energy production. In some embodiments, such plants providehigher yields of ethanol, butanol, other biofuel molecules, and/orsugar-derived co-products per kilogram of plant material, relative tocontrol plants. By providing higher yields at an equivalent or evendecreased cost of production relative to controls, the transgenic plantsdescribed herein improve profitability for farmers and processors aswell as decrease costs to consumers.

Seeds from transgenic plants described herein can be conditioned andbagged in packaging material by means known in the art to form anarticle of manufacture. Packaging material such as paper and cloth arewell known in the art. A package of seed can have a label, e.g., a tagor label secured to the packaging material, a label printed on thepackaging material, or a label inserted within the package, thatdescribes the nature of the seeds therein.

Enhanced salt and/or oxidative stress tolerance gives the opportunity togrow crops in saline or oxidative stress conditions without stuntedgrowth and diminished yields due to salt-induced ion imbalance,disruption of water homeostasis, inhibition of metabolism, damage tomembranes, and/or cell death. The ability to grow plants in saline oroxidative stress conditions would result in an overall expansion ofarable land and increased output of land currently marginally productivedue to elevated salinity or oxidative stress conditions.

Seed or seedling vigor is an important characteristic that can greatlyinfluence successful growth of a plant, such as crop plants. Adverseenvironmental conditions, such as saline and/or oxidative conditions,can affect a plant growth cycle, germination of seeds and seedling vigor(i.e. vitality and strength under such conditions can differentiatebetween successful and failed plant growth). Seedling vigor has oftenbeen defined to comprise the seed properties that determine “thepotential for rapid, uniform emergence and development of normalseedlings under a wide range of field conditions”. Hence, it would beadvantageous to develop plant seeds with increased vigor, particularlyin elevated salinity and/or in oxidative stress conditions.

For example, increased seedling vigor would be advantageous for cerealplants such as rice, maize, wheat, etc. production. For these crops,germination and growth can often be slowed or stopped by salinationand/or oxidation. Genes associated with increased seed vigor undersaline and/or oxidative stress conditions have therefore been sought forproducing improved plant varieties. (Walia et al. (2005) PlantPhysiology 139:822-835).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

VII. EXAMPLES Example 1 Agrobacterium-Mediated Transformation ofArabidopsis

Host Plants and Transgenes: Wild-type Arabidopsis thaliana Wassilewskija(WS) plants were independently transformed with Ti plasmids containingclones encoding polypeptides at SEQ ID NOs: 43, 44, 45, 86, 136, 138,140, 141, 142, 143, 144, and amino acid coordinates 1 to 135 of SEQ IDNO: 140. Examples include Ceres CLONE ID no. 1792354, Ceres SEEDLINE IDno. ME06748, Ceres SEEDLINE ID no. ME08768, Ceres SEEDLINE ID no.ME19173, and Ceres CLONE ID no. 375578. Unless otherwise indicated, eachCeres Clone and/or Seedline derived from a Clone is in the senseorientation relative to either the 35S promoter in a Ti plasmid. A Tiplasmid vector useful for these constructs, CRS 338, contains theCeres-constructed, plant selectable marker gene phosphinothricinacetyltransferase (PAT), which confers herbicide resistance totransformed plants.

Preparation of Soil Mixture: 24 L Sunshine Mix #5 soil (Sun GroHorticulture, Ltd., Bellevue, Wash.) is mixed with 16 L Therm-O-Rockvermiculite (Therm-O-Rock West, Inc., Chandler, Ariz.) in a cement mixerto make a 60:40 soil mixture. To the soil mixture is added 2 TbspMarathon 1% granules (Hummert, Earth City, Mo.), 3 Tbsp OSMOCOTE®14-14-14 (Hummert, Earth City, Mo.) and 1 Tbsp Peters fertilizer20-20-20 (J.R. Peters, Inc., Allentown, Pa.), which are first added to 3gallons of water and then added to the soil and mixed thoroughly.Generally, 4-inch diameter pots are filled with soil mixture. Pots arethen covered with 8-inch squares of nylon netting.

Planting: Using a 60 mL syringe, 35 mL of the seed mixture is aspirated.25 drops are added to each pot. Clear propagation domes are placed ontop of the pots that are then placed under 55% shade cloth andsubirrigated by adding 1 inch of water.

Plant Maintenance: 3 to 4 days after planting, lids and shade cloth areremoved. Plants are watered as needed. After 7-10 days, pots are thinnedto 20 plants per pot using forceps. After 2 weeks, all plants aresubirrigated with Peters fertilizer at a rate of 1 Tsp per gallon ofwater. When bolts are about 5-10 cm long, they are clipped between thefirst node and the base of stem to induce secondary bolts. Dippinginfiltration is performed 6 to 7 days after clipping.

Preparation of Agrobacterium: To 150 mL fresh YEB is added 0.1 mL eachof carbenicillin, spectinomycin and rifampicin (each at 100 mg/ml stockconcentration). Agrobacterium starter blocks are obtained (96-well blockwith Agrobacterium cultures grown to an OD₆₀₀ of approximately 1.0) andinoculated one culture vessel per construct by transferring 1 mL fromappropriate well in the starter block. Cultures are then incubated withshaking at 27° C. Cultures are spun down after attaining an OD₆₀₀ ofapproximately 1.0 (about 24 hours). 200 mL infiltration media is addedto resuspend Agrobacterium pellets. Infiltration media is prepared byadding 2.2 g MS salts, 50 g sucrose, and 5 μL 2 mg/ml benzylaminopurineto 900 ml water.

Dipping Infiltration: The pots are inverted and submerged for 5 minutesso that the aerial portion of the plant is in the Agrobacteriumsuspension. Plants are allowed to grow normally and seed is collected.

Example 2 Saline Condition Screening

Saline condition screening: Screening is routinely performed byhigh-salt agar plate assay and also by high-salt soil assay. Traitsassessed in high-salt conditions include: seedling area, photosynthesisefficiency, salt growth index and regeneration ability.

Seedling area: the total leaf area of a young plant about 2 weeks old.

Photosynthesis efficiency (Fv/Fm): Seedling photosynthetic efficiency,or electron transport via photosystem II, is estimated by therelationship between Fm, the maximum fluorescence signal and thevariable fluorescence, Fv. Here, a reduction in the optimum quantumyield (Fv/Fm) indicates stress, and so can be used to monitor theperformance of transgenic plants compared to non-transgenic plants undersalt stress conditions.

Salt growth index=seedling area x photosynthesis efficiency (Fv/Fm).

Regeneration ability: the ability of a plant to regenerate shoots insaline soil after stems are cut off and the soil is irrigated with 200mM NaCl solution.

Transformant identification: PCR was used to amplify the cDNA insert inone randomly chosen T₂ plant. This PCR product was then sequenced toconfirm the sequence in the plants.

Identification of Tolerant Plant to Salt Stress: A superpool of seedswas screened for transgenic plants that show enhanced tolerance to SA,as detailed below, and high salt. Three independent candidate plantswere sequenced and the transgene sequence matched ME02064.

Assessing Tolerance to Salt Stress: Generally, between four and tenindependently transformed plant lines are selected and qualitativelyevaluated for their tolerance to salt stress in the T₁ generation. Twoor three of the transformed lines that qualitatively show the strongesttolerance to salt stress in the T₁ generation are selected for furtherevaluation in the T₂ and T₃ generations. This evaluation involves sowingseeds from the selected transformed plant lines on MS agar platescontaining either 100 mM or 150 mM NaCl and incubating the seeds for 5to 14 days to allow for germination and growth. For example, for ME02064five T2 events were compared to wild-type Ws for salt stress toleranceon salt plates. Three events, ME02064-01, -03 and -04 were selectedbased on the measurement of seedling area on 36 plants of each event ascompared to the control, Ws. Further evaluation of salt tolerance inME02064-01, -03 and -04 was performed with T₂ and T₃ generations.

Calculating SGI: After germination and growth, seedling area andphotosynthesis efficiency of transformed lines and a wild-type controlare determined. From these measurements, the Salt Growth Index (SGI) iscalculated and compared between wild-type and transformed seedlings. TheSGI calculation is made by multiplying seedling area with photosynthesisefficiency measurements taken from two replicates of 36 seedlings foreach transformed line and a wild-type control and performing a t-test.

Determining Transgene Copy Number: T₂ generation transformed plants aretested on BASTA™ plates in order to determine the transgene copy numberof each transformed line. A BASTA™ resistant:BASTA™ sensitivesegregation ratio of 15:1 generally indicates two copies of thetransgene, and such a segregation ratio of 3:1 generally indicates onecopy of the transgene.

Example 3 Oxidative Stress Conditions Screening

Under normal growth conditions, Arabidopsis rosette contains about 0.5μ/g fresh weight of free SA. In response to stress conditions orpathogen attacks, the free SA levels can reach as high as 10 μg/g freshweight, which is approximately equivalent to 60 μM. The exogenousapplication of 100-500 μM SA to Arabidopsis leaves by spraying is ableto induce strong defense responses without triggering obvious necroticlesion formation. Once the SA concentration increases to 5 mM or above,the cell death in form of necrotic lesions will appear on the sprayedleaves. If SA is applied through growth media, Arabidopsis is moresensitive to SA-induced oxidative stress, probably because of continuousabsorption. The addition of 100-150 μM SA to growth media significantreduces plant growth but does not kills the plants in wild typeArabidopsis Ws. Therefore we use this range of SA to screen for enhancedoxidative stress tolerance.

Salicylic Acid Screening: Screening is routinely performed by agar plateassay using 100 μM or 150 μM exogenous sodium salicylate. Media contains½×MS (Sigma), 150 μM sodium salicylate (Sigma), 0.5 g MES hydrate(Sigma) and 0.7% phytagar (EM Science), adjusted to pH 5.7 using 10NKOH.

To screen superpools, seeds are surface sterilized in 30% bleachsolution for 5 minutes and then rinsed repeatedly with sterile water.Approximately 2500 seeds are sown on media plates in a monolayer at adensity of 850 seeds per plate. Wild-type and positive controls aregrown on comparable plates. Plates are wrapped with vent tape and placedat 4° C. in the dark for three days to stratify. At the end of thistime, plates are transferred to a Conviron growth chamber set at 22° C.,16:8 hour light:dark cycle, 70% humidity with a combination ofincandescent and fluorescent lamps emitting a light intensity of ˜100μEinsteins.

Seedlings are screened daily starting at 6 days. Seedlings that growlarger and stay greener compared to WS control plants are selected aspositive candidates and transferred to soil for recovery and seed set.

Candidate plants are re-screened by placing 36 seeds from each candidatetogether with a WS control on the same sodium salicylate plate. Platesare treated as described above and seedling screening begun after at 4days after germination. Leaf tissue is harvested from confirmed tolerantcandidates for DNA extraction and amplification of the transgene by PCR.

Alternatively, superpool seeds are sown directly on soil and sprayedwith 10 mM SA. Leaf tissue is harvested from tolerant candidate plantsto isolate DNA for PCR amplification of the transgene and subsequentsequencing of the PCR product.

Traits assessed under sodium salicylate conditions include: seedlingarea, photosynthesis efficiency, salicylic acid growth index (SAG) andregeneration ability.

Seedling area: the total leaf area of a young plant about 2 weeks old.

Photosynthesis efficiency (Fv/Fm): Seedling photosynthetic efficiency,or electron transport via photosystem II, is estimated by therelationship between Fm, the maximum fluorescence signal and thevariable fluorescence, Fv. Here, a reduction in the optimum quantumyield (Fv/Fm) indicates stress, and so can be used to monitor theperformance of transgenic plants compared to non-transgenic plants underoxidative stress conditions.

Salicylic Acid Growth (SAG) Index=seedling area (cm²)×photosynthesisefficiency (Fv/Fm).

PCR was used to amplify the cDNA insert in one randomly chosen T₂ plant.This PCR product was then sequenced to confirm the sequence in theplants.

Assessing Tolerance to Oxidative Stress: Initially, All availableindependently transformed T2 plant lines are qualitatively evaluated fortheir tolerance to oxidative stress as compared to wild-type controls.The positive transgenic lines that qualitatively show the strongesttolerance to oxidative stress are selected for further evaluation in theT₂ and T₃ generations using internal non-transgenic segregants ascontrols. This evaluation involves sowing seeds from the selectedtransformed plant lines on MS agar plates containing 100 μM or 150 μMsodium salicylate and incubating the seeds for at least 4 days to allowfor germination and growth and transgene status analysis.

Calculating SAG: After germination and growth, seedling area andphotosynthesis efficiency of transformed lines and a wild-type controlare determined. From these measurements, the Salicylic Acid Growth Index(SAG) is calculated and compared between wild-type and transformedseedlings. The SAG calculation is made by multiplying seedling area withphotosynthesis efficiency measurements taken from two replicates of 36seedlings for each transformed line and a wild-type control andperforming a t-test.

Determining Transgene Copy Number: T₂ generation transformed plants aretested on BASTA™ plates in order to determine the transgene copy numberof each transformed line. A BASTA™ resistant:BASTA™ sensitivesegregation ratio of 15:1 generally indicates two copies of thetransgene, and such a segregation ratio of 3:1 generally indicates onecopy of the transgene.

In some cases, validation is performed using media that is furthersupplemented with 100 uM SNP.

Example 4 ME02064 (Ceres Clone 375578; SEQ ID No.138)

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter and Ceres Clone 375578. Threetransformed lines, ME02064-01 and ME02064-03, ME02064-04, showed thestrongest qualitative tolerance to salt stress in a prevalidation assay(Table 4-1). Their tolerance to 150 mM NaCl was further evaluated in avalidation assay for two generations. Segregation ratios (BASTA™resistant: BASTA™ sensitive) indicated ME02064-01 and ME02064-03,ME02064-04 transformed lines each carry one copy of the transgene.

TABLE 4-1 Prevalidation assay of ME02064 salt tolerance as compared towild-type Ws Ws ME02064- ME02064- Wild-type ME02064-01 ME02064-02ME02064-03 04 05 Mean* 0.0359 0.0435 0.0346 0.0441 0.0438 0.0305Standard Error 0.0016 0.0048 0.004 0.0041 0.0035 0.0019 *Averageseedling area of 36 plants grown on MS agar plates containing 150 mMNaCl for 14 days

When grown on MS agar plates containing 150 mM NaCl, ME02064-01 andME02064-03, ME02064-04 transgenic plants showed significantly greaterseedling area and SGI relative to non-transgenic plants. As shown inTable 4-2, the T2-generation SGI value for ME02064-01 seedlingsincreased by 110% while ME02064-03 seedlings increased by 131% andME02064-04 seedlings increased by 72% compared to non-transgenic controlseedlings. In the T₃ generation, the SGI increase was 43% forME02064-01, 47% for ME02064-03, and 64% for ME02064-04. The differencesbetween transgenic and non-transgenic seedlings are statisticallysignificant, and clearly demonstrate that the enhanced tolerance to saltstress was a result of the ectopic expression of Ceres Clone 375578 inthe ME02064 transformant lines.

TABLE 4-2 Validation assay of ME02064 on salt tolerance in twogenerations SGI of pooled non- % of SGI* of transgenics transgenicst-Test SGI ME Events Avg SE N Avg SE N t-value t_(0.05) increaseME02064-01-T₂ 2.057 0.249 12 0.977 0.205 17 3.35 1.70 110.5ME02064-03-T₂ 2.237 0.371 5 0.968 0.140 24 3.20 1.70 131.1 ME02064-04-T₂1.810 0.146 14 1.055 0.135 13 3.81 1.70 71.6 ME02064-01-T₃ 2.438 0.17021 1.708 0.289 9 2.18 1.70 42.7 ME02064-03-T₃ 2.837 0.257 20 1.927 0.27114 2.43 1.70 47.2 ME02064-04-T₃ 2.770 0.318 16 1.688 0.188 19 2.93 1.7064.1 *SGI (Salt Growth Index) = seedling area x Fv/Fm (photosynthesisefficiency)Summary of Results:

-   -   Ectopic expression of Ceres Clone 375578 under the control of        the 35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.

Example 5 ME03140; Clone 375578; SEQ ID No.142

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to Ceres Clone375578 (SEQ ID NO: 142), and five transgenic lines, ME03140-01,ME03140-02, ME03140-03, ME03140-04 and ME03140-05 were investigated fortolerance to salt stress. When grown on MS agar plates containing 150 mMNaCl, these transgenic lines showed increased tolerance to salt stressin quantitative assays as compared to non-transgenic control seedlings.

When grown on MS agar plates containing 150 mM NaCl, ME03140-01,ME03140-02, ME03140-03, ME03140-04 and ME03140-05 transgenic plantsshowed significantly greater seedling area and SGI relative tonon-transgenic plants. As shown in Table 5, the T2-generation SGI valuefor ME03140-01 seedlings increased 102.18%, ME03140-02 seedlingsincreased 60.78%, ME03140-03 seedlings increased 120.32%, ME03140-04seedlings increased 45.07% and ME03140-05 seedlings increased 90.53% ascompared to non-transgenic control seedlings. The differences in SGIvalues between transgenic and non-transgenic seedlings havestatistically significant P values for all transgenic lines, and thesequantitative experiments clearly demonstrate that ectopic expression ofCeres Clone 375578 confers enhanced tolerance to salt stress intransgenic seedlings.

TABLE 5 Validation assay of ME03140 salt stress tolerance in onegeneration SGI of pooled non- ME SGI* of transgenics transgenics P % ofEvents Avg SE N Avg SE N value SGI increase ME03140-01-T₂ 4.340.590403017 17 2.15 0.478695 26 3.10E−03 102.18% ME03140-02-T₂ 4.090.395692005 18 2.54 0.367281 28 3.22E−03  60.78% ME03140-03-T₂ 4.030.646365854 12 1.83 0.397508 36 2.86E−03 120.32% ME03140-04-T₂ 4.860.534320049 17 3.35 0.446161 36 1.74E−02  45.07% ME03140-05-T₂ 4.310.5237326 25 2.26 0.665646 20 9.91E−03  90.53% *SGI (Salt Growth Index)= seedling area x Fv/Fm (photosynthesis efficiency)Summary of Results:

-   -   Ectopic expression of Ceres Clone 375578 under the control of        the 35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.

Example 6 ME08732; Clone 560066; SEQ ID No. 44

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to Ceres Clone560066 (SEQ ID NO: 44), and three transgenic lines, ME08732-01,ME08732-02 and ME08732-03, were investigated for tolerance to saltstress. When grown on MS agar plates containing 150 mM NaCl, thesetransgenic lines showed increased tolerance to salt stress inquantitative assays as compared to non-transgenic control seedlings.

When grown on MS agar plates containing 150 mM NaCl, ME08732-01,ME08732-02 and ME08732-03 transgenic plants showed significantly greaterseedling area and SGI relative to non-transgenic plants. As shown inTable 6, the T2-generation SGI value for ME08732-01 seedlings increased88.35%, ME08732-02 seedlings increased 41.72% and ME08732-03 seedlingsincreased 26.23%, as compared to non-transgenic control seedlings. Thedifferences in SGI values between transgenic and non-transgenicseedlings have statistically significant P values for ME08732-01 andME08732-02 transgenic lines, and these quantitative experiments clearlydemonstrate that ectopic expression of Ceres Clone 560066 confersenhanced tolerance to salt stress in transgenic seedlings.

TABLE 6 Validation assay of ME08732 salt stress tolerance in onegeneration SGI of pooled ME SGI* of transgenics non-transgenics P % ofEvents Avg SE N Avg SE N value SGI increase ME08732-01-T₂ 4.070.164301729 24 2.16 0.472565 14 2.57E−04 88.35% ME08732-02-T₂ 3.420.391450599 21 2.41 0.336042 26 2.86E−02 41.72% ME08732-03-T₂ 4.710.566761111 10 3.73 0.285925 52 6.44E−02 26.23% *SGI (Salt Growth Index)= seedling area x Fv/Fm (photosynthesis efficiency)Summary of Results:

-   -   Ectopic expression of Ceres Clone 560066 under the control of        the 35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.

Example 7 ME08768; Clone 539458; SEQ ID No.86

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to Ceres Clone539458 (SEQ ID NO: 86), and five transgenic lines, ME08768-01,ME08768-02, ME08768-03, ME08768-04 and ME08768-05, were investigated fortolerance to salt stress. When grown on MS agar plates containing 150 mMNaCl, these transgenic lines showed increased tolerance to salt stressin quantitative assays as compared to non-transgenic control seedlings.

When grown on MS agar plates containing 150 mM NaCl, ME08768-01,ME08768-02, ME08768-03, ME08768-04 and ME08768-05 transgenic plantsshowed significantly greater seedling area and SGI relative tonon-transgenic plants. As shown in Table 7, the T2-generation SGI valuefor ME08768-01 seedlings increased 80.04%, ME008768-02 seedlingsincreased 111.63%, ME008768-03 seedlings increased 22.62%, ME008768-04seedlings increased 115.40% and ME008768-05 seedlings increased 74.41%as compared to non-transgenic control seedlings. The differences in SGIvalues between transgenic and non-transgenic seedlings havestatistically significant P values for ME08768-01, ME08768-02,ME08768-04 and ME08768-05 transgenic lines, and these quantitativeexperiments clearly demonstrate that ectopic expression of Ceres Clone539458 confers enhanced tolerance to salt stress in transgenicseedlings.

TABLE 7 Validation assay of ME08768 salt stress tolerance in onegeneration SGI of pooled non- % of ME SGI* of transgenics transgenics PSGI Events Avg SE N Avg SE N value increase ME08768-01-T₂ 14.481.254125111 20 8.04 1.321838 26 4.91E−04  80.04% ME08768-02-T₂ 11.090.822117225 20 5.24 0.751908 32 1.55E−06 111.63% ME08768-03-T₂ 13.721.676864172 21 11.19 1.57188 30 0.1380406  22.62% ME08768-04-T₂ 14.821.3958585 16 6.88 0.777162 40 3.58E−06 115.40% ME08768-05-T₂ 10.021.365308 13 5.75 0.751134 38 4.23E−03  74.41% *SGI (Salt Growth Index) =seedling area x Fv/Fm (photosynthesis efficiency)Summary of Results:

-   -   Ectopic expression of Ceres Clone 539458 under the control of        the 35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.

Example 8 ME10681; Clone 335348 SEQ ID No.141

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to Ceres Clone335348 (SEQ ID NO: 141), and six transgenic lines, ME10681-01-T₂,ME10681-01-T3, ME10681-02-T₂, ME10681-02-T3, ME10681-04-T₂ andME10681-05-T₂, were investigated for tolerance to salt stress. Whengrown on MS agar plates containing 150 mM NaCl, these transgenic linesshowed increased tolerance to salt stress in quantitative assays ascompared to non-transgenic control seedlings.

When grown on MS agar plates containing 150 mM NaCl, ME10681-01-T₂,ME10681-01-T₃, ME10681-02-T₂, ME10681-02-T₃, ME10681-04-T₂ andME10681-05-T₂ transgenic plants showed significantly greater seedlingarea and SGI relative to non-transgenic plants. As shown in Table 8, theT2-generation SGI value for ME010681-01-T₂ seedlings increased 39.17%,ME010681-01-T₃ seedlings increased 19.77%%, ME10681-02-T₂ seedlingsincreased 119.17%, ME10681-02-T₃ seedlings increased 6.21%,ME010681-04-T₂ seedlings increased 113.51% and ME010681-05-T₂ seedlingsincreased 103.98%, as compared to non-transgenic control seedlings. Thedifferences in SGI values between transgenic and non-transgenicseedlings have statistically significant P values for ME10681-01-T₃,ME10681-02-T₂, ME10681-04-T₂ and ME10681-05-T₂ transgenic lines, andthese quantitative experiments clearly demonstrate that ectopicexpression of Ceres Clone 335348 confers enhanced tolerance to saltstress in transgenic seedlings.

TABLE 8 Validation assay of ME10681 salt stress tolerance in twogenerations SGI of pooled non- ME SGI* of transgenics transgenics % ofEvents Avg SE N Avg SE N P value SGI increase ME10681-01-T₂ 3.870.683711333 9 2.78 0.302501 48 7.54E−02  39.17% ME10681-01-T₃ 4.70.31544415 23 3.93 0.3015141 43 3.99E−02  19.77% ME10681-02-T₂ 4.130.3353564 25 1.89 0.3969 22 4.16E−05 119.17% ME10681-02-T₃ 3.650.258400663 31 3.44 0.3060094 34 0.2980488  6.21% ME10681-04-T₂ 6.220.478672159 12 2.91 0.39405 30 2.04E−06 113.51% ME10681-05-T₂ 5.250.391550037 20 2.57 0.4265902 30 1.44E−05 103.98% *SGI (Salt GrowthIndex) = seedling area x Fv/Fm (photosynthesis efficiency)Summary of Results:

-   -   Ectopic expression of Ceres Clone 335348 under the control of        the 35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.

Example 9 ME18973; Ceres cDNA ID 23457556; SEQ ID No.43

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to Ceres cDNA ID23457556 (SEQ ID NO: 43), and six transgenic lines, ME18973-01-T₂,ME18973-02-T₂, ME18973-02-01-T₃, ME18973-03-T₂, ME18973-05-T₂ andME18973-05-03-T₃ were investigated for tolerance to salt stress. Whengrown on MS agar plates containing 150 mM NaCl, these transgenic linesshowed increased tolerance to salt stress in quantitative assays ascompared to non-transgenic control seedlings.

When grown on MS agar plates containing 150 mM NaCl, ME18973-01,ME18973-02-T₂, ME18973-02-01-T₃, ME18973-03-T₂, ME18973-05-T₂ andME18973-05-03-T₃ transgenic plants showed significantly greater seedlingarea and SGI relative to non-transgenic plants. As shown in Table 9, theT2 & T3-generation SGI value for ME018973-01-T₂ seedlings increased230.01%, ME18973-02-T₂ seedlings increased 22.44%, ME18973-02-01-T₃seedlings increased 14.96%, ME18973-05-T₂ seedlings increased 16.12% andME18973-05-03-T₃ seedlings increased 13.97%, as compared tonon-transgenic control seedlings. The differences in SGI values betweentransgenic and non-transgenic seedlings have statistically significant Pvalues for the ME18973 transgenic lines, and these quantitativeexperiments clearly demonstrate that ectopic expression of Ceres cDNA ID23457556 results in enhanced tolerance to salt stress in transgenicseedlings.

TABLE 9 Validation assay of ME18973 salt stress tolerance in twogenerations SGI of pooled non- % of ME SGI* of transgenics transgenicsSGI Events Avg SE N Avg SE N P value increase ME18973-01-T₂ 4.410.253654648 26 1.34 0.367022 18 1.03E−08 230.01% ME18973-02-T₂ 4.470.373604899 27 3.65 0.526316 18 0.1058348  22.44% ME18973-02-01-T₃ 4.820.205971746 44 4.19 0.3832982 25 7.71E−02  14.96% ME18973-05-T₂ 4.74 0 14.09 0.503725 26 0.160517  16.12% ME18973-05-03-T₃ 4.38 0.233610226 323.84 0.503725 37 6.89E−02  13.97% *SGI (Salt Growth Index) = seedlingarea x Fv/Fm (photosynthesis efficiency)Summary of Results:

-   -   Ectopic expression of Ceres cDNA ID 23457556 under the control        of the 35S promoter enhances tolerance to salt stress that        causes necrotic lesions and stunted growth in wild-type Ws        seedlings.

Example 10 ME19657; cDNA ID 23621377; SEQ ID No.45

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to Ceres cDNA ID23621377 (SEQ ID NO: 45), and two transgenic lines, ME19657-01-T₂,ME19657-01-05-T₃, ME19657-01-08-T₃, ME19657-02-T₂, ME19657-03-T₂,ME19657-04-T₂ and ME19657-04-01-T₃, were investigated for tolerance tosalt stress. When grown on MS agar plates containing 150 mM NaCl, thesetransgenic lines showed increase tolerance to salt stress inquantitative assays as compared to non-transgenic control seedlings.

When grown on MS agar plates containing 150 mM NaCl, ME19657-01-T₂,ME19657-01-05-T₃, ME19657-01-08-T₃, ME19657-02-T₂, ME19657-03-T₂,ME19657-04-T₂ and ME19657-04-01-T₃ transgenic plants showedsignificantly greater seedling area and SGI relative to non-transgenicplants. As shown in Table 10, the T2 & T3-generation SGI value forME19657-01-T₂ seedlings increased 82.29%, ME19657-01-05-T₃ seedlingsincreased 82.29%, ME19657-01-08-T₃ seedlings increased 21.90%,ME19657-02-T₂ seedlings increased 39.50%, ME19657-03-T₂ seedlingsincreased 98.28%, and ME19657-04-T₂ seedlings increased 4.38% andME19657-04-01-T₂ seedlings increased 7.44%, as compared tonon-transgenic control seedlings. The differences in SGI values betweentransgenic and non-transgenic seedlings have statistically significant Pvalues for ME19657-01-T₂, ME19657-01-05-T₃, ME19657-01-08-T₃,ME19657-02-T₂ and ME19657-03-T₂ transgenic lines, and these quantitativeexperiments clearly demonstrate that ectopic expression of Ceres cDNA ID23621377 results in enhanced tolerance to salt stress in transgenicseedlings.

TABLE 10 Validation assay of ME19657 salt stress tolerance in twogenerations SGI of pooled non- % of ME SGI* of transgenics transgenicsSGI Events Avg SE N Avg SE N P value increase ME19657-01-T₂ 4.540.311964078 21 2.49 0.539972 15 5.62E−05 82.29% ME19657-01-05-T₃ 0.70.311964078 21 0.7 0.5399721 15 1.18E−03 82.29% ME19657-01-08-T₃ 5.40.278520121 27 4.43 0.3061552 36 1.18E−03 21.90% ME19657-02-T₂ 3.970.32089576 23 2.84 0.527849 18 0.0111868 39.50% ME19657-03-T₂ 4.790.313786256 22 2.41 0.299954 22 3.83E−02 98.28% ME19657-04-T₂ 3.670.341681304 15 3.52 0.324049 40 1.15E−06  4.38% ME19657-04-01-T₃ 4.560.495154 9 4.25 0.3487774 37 0.3723989  7.44% *SGI (Salt Growth Index) =seedling area x Fv/Fm (photosynthesis efficiency)Summary of Results:

-   -   Ectopic expression of Ceres cDNA ID 23621377 under the control        of the 35S promoter enhances tolerance to salt stress that        causes necrotic lesions and stunted growth in wild-type Ws        seedlings.

Example 11 ME24076; Clone 229668; SEQ ID No. 143

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to Ceres Clone:229668 (SEQ ID NO: 143), and two transgenic lines, ME24076-01 andME24076-02, were investigated for tolerance to salt stress. When grownon MS agar plates containing 150 mM NaCl, these transgenic lines showedincrease tolerance to salt stress in quantitative assays as compared tonon-transgenic control seedlings.

When grown on MS agar plates containing 150 mM NaCl, only ME024076-01-T₂and transgenic plants showed significantly greater seedling area and SGIrelative to non-transgenic plants. As shown in Table 11, theT2-generation SGI value for ME24076-01-T₂ seedlings increased 65.57% andME24076-02-T₂ seedlings decreased by 1.12%, as compared tonon-transgenic control seedlings. The differences in SGI values betweentransgenic and non-transgenic seedlings have statistically significant Pvalues for transgenic line ME24076-01, and these quantitativeexperiments clearly demonstrate that ectopic expression of Ceres Clone229668 results in enhanced tolerance to salt stress in transgenicseedlings.

TABLE 11 Validation assay of ME24076 salt stress tolerance in onegeneration SGI of pooled non- % of ME SGI* of transgenics transgenicsSGI Events Avg SE N Avg SE N P value increase ME24076-01-T₂ 11.180.924279499 17 6.75 0.9761984 32 9.45E−04 65.57% ME24076-02-T₂  0.70.082529059 10 0.7  0.0506174 48 0.4675565 −1.12% *SGI (Salt GrowthIndex) = seedling area x Fv/Fm (photosynthesis efficiency)Summary of Results:

-   -   Ectopic expression of Ceres Clone 229668 under the control of        the 35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.

Example 12 ME24217; Clone 375578; SEQ ID No. 144

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to Ceres Clone375578 (SEQ ID NO: 144), and two transgenic lines, ME24217-07-T₂ andME24217-09-T₂, were investigated for tolerance to salt stress. Whengrown on MS agar plates containing 150 mM NaCl, these transgenic linesshowed increase tolerance to salt stress in quantitative assays ascompared to non-transgenic control seedlings.

When grown on MS agar plates containing 150 mM NaCl, ME24217-07-T₂ andME24217-09-T₂ transgenic plants showed significantly greater seedlingarea and SGI relative to non-transgenic plants. As shown in Table 12,the T2-generation SGI value for ME24217-07 seedlings increased 30.41%and ME24217-09 seedlings increased 134.46%, as compared tonon-transgenic control seedlings. The differences in SGI values betweentransgenic and non-transgenic seedlings have statistically significant Pvalues for ME24217-07-T₂ and ME24217-09-T₂ transgenic lines, and thesequantitative experiments clearly demonstrate that ectopic expression ofCeres Clone 375578 results in enhanced tolerance to salt stress intransgenic seedlings.

TABLE 12 Validation assay of ME24217salt stress tolerance in onegeneration SGI of pooled non- ME SGI* of transgenics transgenics P % ofEvents Avg SE N Avg SE N value SGI increase ME24217-07-T₂ 4.690.413823734 20 3.6 0.4284669 30 3.62E−02  30.41% ME24217-09-T₂ 4.920.446345081 22 2.1 0.506974 22 7.20E−05 134.46% *SGI (Salt Growth Index)= seedling area x Fv/Fm (photosynthesis efficiency)Summary of Results:

-   -   Ectopic expression of Ceres Clone 375578 under the control of        the 35S promoter enhances tolerance to salt stress that causes        necrotic lesions and stunted growth in wild-type Ws seedlings.

Example 13 ME02064C; Clone 375578C; SEQ ID No. 140

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to Ceres Clone375578 (SEQ ID NO: 140), and six transgenic lines, ME02064C-01-T₂,ME02064C-02-T₂, ME02064C-03-T₂, ME02064C-04-T₂, ME02064C-05-T₂ andME02064C-06-T₂ were investigated for tolerance to salt stress. Whengrown on MS agar plates containing 150 mM NaCl, most of these transgeniclines did not show tolerance to salt stress in quantitative assays ascompared to non-transgenic control seedlings.

Table 13 shows that, when grown on MS agar plates containing 100 mMNaCl, the T2-generation SGI value for: ME02064C-01-T₂ seedlings ascompared to non-transgenic control seedlings was 0.55%; ME02064C-02-T₂seedlings as compared to non-transgenic control seedlings was 1.31%;ME02064C-03-T₂ seedlings as compared to non-transgenic control seedlingswas 9.67%; ME02064C-04-T₂ seedlings as compared to non-transgeniccontrol seedlings was −7.78%; ME02064C-05-T₂ seedlings as compared tonon-transgenic control seedlings was −15.77%; and ME02064C-06-T₂seedlings as compared to non-transgenic control seedlings 17.78%.

TABLE 13 Validation assay of ME02064C salt stress tolerance in onegeneration SGI of pooled non- % of ME SGI* of transgenics transgenicsSGI Events Avg SE N Avg SE N P value increase ME02064C-01-T₂ 10.890.735174679 33 10.83 0.707901 34 0.4769106  0.55% ME02064C-02-T₂ 10.70.595225094 50 10.56 0.971548 21 0.4517289  1.31% ME02064C-03-T₂  9.390.582009053 48  8.56 0.958475 23 0.2314441  9.67% ME02064C-04-T₂ 10.660.555387069 51 11.56 1.046386 21 0.2252269  −7.78% ME02064C-05-T₂ 10.840.60377588  48 12.87 0.839921 24 2.68E−02 −15.77% ME02064C-06-T₂ 12.550.608556025 44 10.65 0.764179 28 2.83E−02  17.78% *SGI (Salt GrowthIndex) = seedling area x Fv/Fm (photosynthesis efficiency)Summary of Results:

-   -   Ectopic expression of Ceres Clone 375578 under the control of        the 35S might not promote enhances tolerance to salt stress that        causes necrotic lesions and stunted growth in wild-type Ws        seedlings.

Example 14 ME02064P1; Clone 375578P1-Amino Acids 1 to 135 of SEQ ID No.140

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to a nucleic acidencoding Ceres Clone 375578P1 (amino acids 1 to 135 of SEQ ID NO: 140),a 3′ truncation variant of Ceres Clone 375578 described above inExample 1. Five transgenic lines, ME02064P1-03-T₂, ME02064P1-07-T₂,ME02064P1-09-T₂, ME02064P1-10-T₂ and ME02064P1-15-T₂ were investigatedfor tolerance to salt stress. All five of these transgenic lines showedtolerance to salt stress in quantitative assays as compared tonon-transgenic control seedlings. As shown in Table 10, theT2-generation SGI value for ME02064P1 seedlings increased by 32.57%,89.52%, 66.84%, 25.43%, 36.95%, compared to non-transgenic controlseedlings.

When grown on MS agar plates containing 150 mM NaCl, ME02064P1-03,ME02064P1-07, ME02064P1-09, ME02064P1-10 and ME02064P1-15 transgenicplants showed significantly greater seedling area and SGI relative tonon-transgenic plants. As shown in Table 14, the T2-generation SGI valuefor ME02064P1-03 seedlings increased 32.57%, ME02064P1-07 seedlingsincreased 89.52%, ME02064P1-09 seedlings increased 66.84%, ME02064P1-10seedlings increased 25.43% and ME02064P1-15 seedlings increased 36.95%as compared to non-transgenic control seedlings. The differences in SGIvalues between transgenic and non-transgenic seedlings havestatistically significant under P values for transgenic linesME02064P1-03-T₂, ME02064P1-07-T₂, ME02064P1-09-T₂, ME02064P1-10-T₂ andME02064P1-15-T₂, and these quantitative experiments clearly demonstratethat ectopic expression of Ceres Clone 37558P1 results in enhancedtolerance to salt stress in transgenic seedlings.

TABLE 14 Validation assay of ME02064P1 salt stress tolerance in onegeneration SGI of pooled non- % of SGI* of transgenics transgenics P SGIME Events Avg SE N Avg SE N value increase ME02064P1-03-T₂ 10.760.507929031 47 8.12 0.925474 25 7.29E−03 32.57% ME02064P1-07-T₂ 13.260.561088966 54 7 1.165372 16 3.87E−06 89.52% ME02064P1-09-T₂ 12.230.654850534 54 7.33 1.141553 17 1.99E−04 66.84% ME02064P1-10-T₂ 15.630.570291003 40 12.46 0.845552 32 1.36E−03 25.43% ME02064P1-15-T₂ 11.840.607966 42 8.64 0.959856 30 3.20E−03 36.95% *SGI (Salt Growth Index) =seedling area x Fv/Fm (photosynthesis efficiency)Summary of Results:

-   -   Ectopic expression of Clone 375587P1 under the control of the        35S promoter enhances tolerance to salt stress.

Example 15 ME02064P2; Clone 375578P2-Amino Acids 188 to 498 of SEQ IDNo.140

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter and a nucleic acid encoding CeresClone 375578P2 (amino acids 188 to 498 of SEQ ID NO: 140), a 5′truncation variant of Ceres Clone 375578 described above in Example 1.Eight ME02064P2 transgenic lines were investigated for tolerance tosalt. Four transgenic lines, ME02064P2-01-T₂, ME02064CP2-04-T₂,ME02064P2-05-T₂, ME02064P2-06-T₂ ME02064P2-07-T₂, ME02064P2-T₂-08 andME02064P2-09-T₂ did show statistically significant salt tolerance inquantitative assays as compared to non-transgenic control seedlings; andone transgenic lines, ME02064P2-10-T₂, showed statistically significantreduction in salt tolerance as compared to non-transgenic controlseedlings.

Table 15 shows that, when grown on MS agar plates containing 100 mMNaCl, the T2-generation SGI value for: ME02064P2-01-T₂ seedlings ascompared to non-transgenic control seedlings was 1.62%, ME02064P2-04-T₂seedlings as compared to non-transgenic control seedlings was 20.31%,ME02064P2-05-T₂ seedlings as compared to non-transgenic controlseedlings was 31.24%, ME02064P2-06-T₂ seedlings as compared tonon-transgenic control seedlings was 41.14%, ME02064P2-07-T₂ seedlingsas compared to non-transgenic control seedlings was 15.91%,ME02064P2-08-T₂ seedlings as compared to non-transgenic controlseedlings was 40.82%, ME02064P2-09-T₂ seedlings as compared tonon-transgenic control seedlings was 135.79%, and ME02064P2-10-T₂ was−12.36% as compared to non-transgenic control seedlings.

When grown on MS agar plates containing 100 mM NaCl, ME02064P2-01-T₂,ME02064P2-04-T₂, ME02064P2-05-T₂, ME02064P2-06-T₂, ME02064P2-07-T₂,ME02064P2-08-T₂ and ME02064P2-09-T₂ transgenic plants showedsignificantly greater seedling area and SGI relative to non-transgenicplants. However as shown in Table 3, the T2-generation SGI value forME02064P2-10-T₂ seedlings showed a decrease in SGI compared tonon-transgenic control seedlings.

TABLE 15 Validation assay of ME02064P2 on salt tolerance in onegeneration SGI of pooled non- % of SGI* of transgenics transgenics SGIME Events Avg SE N Avg SE N P value increase ME02064P2-01-T₂ 9.840.687493743 53 9.68 1.261045 19 0.4567634  1.62% ME02064P2-04-T₂ 5.20.558723451 47 4.32 0.560634 25 0.1357713  20.31% ME02064P2-05-T₂ 8.420.714218299 45 6.41 0.623421 27 0.0190578  31.24% ME02064P2-06-T₂ 8.560.515029349 48 6.07 0.654098 24 1.88E−03  41.14% ME02064P2-07-T₂ 12.30.647077232 47 10.61 0.8768 25 6.29E−02  15.91% ME02064P2-08-T₂ 9.160.724681422 37 6.51 0.73405 35 6.08E−03  40.82% ME02064P2-09-T₂ 5.720.489863069 47 2.43 0.182583 24 1.19E−08 135.79% ME02064P2-10-T₂ 9.320.908174851 21 10.63 0.70877 51 0.1289273  12.36% *SGI (Salt GrowthIndex) = seedling area x Fv/Fm (photosynthesis efficiencySummary of Results:

-   -   Ectopic expression of Clone 375587P2 under the control of the        35S promoter enhances tolerance to salt stress.    -   Ceres Clone 375578P2 retains the α-β domains of Ceres Clone        375578 located within amino acid residues 137-157 of SEQ ID        NO: 140) but does not retain the σ-Γ domains of Ceres Clone        375578 of SEQ ID NO: 140.

Example 16 ME10681; Clone 335348 SEQ ID No. 141

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to Ceres cDNA335348 (SEQ ID NO: 141). Wildtype Ws seedlings showed necrotic lesionsand stunted growth on plates containing 100 or 150 μM SA, whereas thetransgenic plants showed significantly better growth.

Three transformed lines, ME10681-01, ME10681-02 and ME10681-05, werequantitatively studied by growth on MS agar plates containing 100 μM SA.After 14 days, plates were scanned using an EPSON color scanner orfluorescence scanner and SAGI calculated for each plant. The data issummarized in Table 16.

When grown on MS agar plates containing 100 μM SA, ME10681-02-T₂ andME10681-05-T₂ transgenic plants showed significantly increased seedlingarea and SAGI relative to non-transgenic plants. However ME10681-01-T₂showed a slight decrease in SAGI relative to non-transgenic plants. Asshown in Table 12, the T₂ generation SAGI value for ME10681-01-T₂,ME10681-02-T₂ and ME10681-05-T₂ seedlings was −3.29%, 17.65% and 51.84%,respectively. The differences between transgenic and non-transgenicseedlings have statistically significant P values for linesME10681-02-T₂ and ME10681-05-T₂, and clearly demonstrate enhancedtolerance to oxidative stress is a result of the ectopic expression ofCeres cDNA 36505846 in the ME10681 transformant lines.

TABLE 16 Salicylic acid validation assay of ME10681 in one generationSeedling area of Seedling area of pooled non- % transgenics transgenicsSeedling ME Events Avg SE N Avg SE N P value area increase ME10681-01-T₂0.56 0.096445 18 0.58 0.061856 53 0.434159 −3.29% ME10681-02-T₂ 0.670.06042 38 0.38 0.079644 32 0.002198 17.65% ME10681-05-T₂ 0.68 0.07227143 0.45 0.108539 25 0.039761 51.84%Summary of Results:

-   -   In sum, ectopic expression of Ceres Clone 335348 under the        control of the 35S promoter enhances oxidative stress tolerance        that causes necrotic lesions and stunted growth in wild-type WS        seedlings.

Example 17 ME24091; Clone 106263; SEQ ID No. 136

Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Tiplasmid carrying the 35S promoter operatively linked to Ceres cDNA016263 (SEQ ID NO: 135). Wildtype Ws seedlings showed necrotic lesionsand stunted growth on plates containing 100 or 150 μM SA, whereas thetransgenic plants showed significantly better growth.

Ten transformed lines, ME24091-01-T₂, ME24091-02-T₂, ME24091-03-T₂,ME24091-04-T₂ ME24091-05-T₂, ME24091-06-T₂ ME24091-07-T₂, ME24091-08-T₂,ME24091-09-T₂ and ME24091-10-T₂, were quantitatively studied by growthon MS agar plates containing 100 μM SA. After 14 days, plates werescanned using an EPSON color scanner or fluorescence scanner and SAGIcalculated for each plant.

When grown on MS agar plates containing 100 μM SA, ME24091-01-T₂,ME24091-02-T₂, ME24091-03-T₂, ME24091-04-01-T₃, ME24091-04-T₂,ME24091-05-01-T₃, ME24091-05-T₂, ME24091-06-01, ME24091-06,ME24091-07-01, ME24091-07, ME24091-08-01, ME24091-08, ME24091-09-01,ME24091-09, ME24091-10-01 and ME24091-10 transgenic plants showedsignificantly increased seedling area and SAGI relative tonon-transgenic plants. As shown in Table 17, the T₂ generation SAGIvalue for ME24091-01, ME24091-02, ME24091-03, ME24091-04 ME24091-05,ME24091-06 ME24091-07, ME24091-08, ME24091-09 and ME24091-10 seedlingsincreased by 119.47%, 198.00% and 133.67%, 241.50%, 143.70% and 248.12%,186.59%, 188.86%, 285.42% and 180.46% respectively. The differencesbetween transgenic and non-transgenic seedlings have statisticallysignificant P values for transgenic lines ME24091-01, ME24091-02,ME24091-03, ME24091-04-01, ME24091-04 ME24091-05-01, ME24091-05,ME24091-06-01, WE24091-06, ME24091-07-01, ME24091-07, ME24091-08,ME24091-09-01, ME24091-09, and ME24091-10, and clearly demonstrate thatthe enhanced tolerance to oxidative stress is a result of the ectopicexpression of Ceres Clone 106263 in the ME24091 transformant lines.

TABLE 17 Salicylic acid validation assay of ME24091 in two generationsSeedling area of Seedling area of pooled % Seedling transgenicsnon-transgenics area ME Events Avg SE N Avg SE N P value increaseME24091-01-T₂ 0.69 0.055882059 29 0.58 0.070002209 38 0.105475324 19.47% ME24091-02-T₂ 0.44 0.050576014 41 0.22 0.054717602 270.002577564  98.00% ME24091-03-T₂ 0.58 0.054269056 43 0.44 0.08571522426 0.076183067  33.67% ME24091-04-T₂ 0.54 0.050859903 45 0.220.077668008 19 0.000634704 141.50% ME24091-04-01-T₃ 0.39 0.07715765 200.24 0.07271465 20 0.081950663  61.93% ME24091-05-T₂ 0.55 0.048581793 420.38 0.072915009 27 0.029849118  43.70% ME24091-05-01-T₃ 0.380.068463201 21 0.15 0.05109963 30 0.005958129 144.90% ME24091-06-T₂ 0.710.049360913 39 0.29 0.063969074 23 1.13831E−06 148.12% ME24091-06-01-T₂0.49 0.073404661 19 0.22 0.063271768 22 0.004691952 118.19%ME24091-07-T₂ 0.69 0.054095931 37 0.37 0.07390372 25 0.000414138  86.59%ME24091-07-01-T₃ 0.49 0.052850446 33 0.19 0.049649799 22  5.3153E−05162.61% ME24091-08-T₂ 0.64 0.059981819 24 0.34 0.071776729 23 0.00111815 88.86% ME24091-08-01-T₃ 0.44 0.050181996 27 0.40 0.074557785 260.306877156  11.48% ME24091-09-T₂ 0.81 0.056031311 38 0.29 0.06740306522 5.88685E−08 185.42% ME24091-09-01-T₃ 0.45 0.055439617 36 0.280.05131548 31 0.0116714  62.95% ME24091-10-T₂ 0.56 0.048643058 39 0.310.062146975 29 0.001240527  80.46% ME24091-10-01-T₃ 0.36 0.051198395 310.26 0.066281225 22 0.114418402  39.44%Summary of Results:

-   -   In sum, ectopic expression of Ceres cDNA Clone 106263 under the        control of the 35S promoter enhances oxidative stress tolerance        that causes necrotic lesions and stunted growth in wild-type Ws        seedlings.

Example 18 Determination of Functional Homologs by Reciprocal BLAST

A candidate sequence was considered a functional homolog of a referencesequence if the candidate and reference sequences encoded proteinshaving a similar function and/or activity. A process known as ReciprocalBLAST (Rivera et al., Proc. Natl. Acad. Sci. USA, 95:6239-6244 (1998))was used to identify potential functional homolog sequences fromdatabases consisting of all available public and proprietary peptidesequences, including NR from NCBI and peptide translations from Ceresclones.

Before starting a Reciprocal BLAST process, a specific referencepolypeptide was searched against all peptides from its source speciesusing BLAST in order to identify polypeptides having BLAST sequenceidentity of 80% or greater to the reference polypeptide and an alignmentlength of 85% or greater along the shorter sequence in the alignment.The reference polypeptide and any of the aforementioned identifiedpolypeptides were designated as a cluster.

The BLASTP version 2.0 program from Washington University at SaintLouis, Mo., USA was used to determine BLAST sequence identity andE-value. The BLASTP version 2.0 program includes the followingparameters: 1) an E-value cutoff of 1.0e-5; 2) a word size of 5; and 3)the -postsw option. The BLAST sequence identity was calculated based onthe alignment of the first BLAST HSP (High-scoring Segment Pairs) of theidentified potential functional homolog sequence with a specificreference polypeptide. The number of identically matched residues in theBLAST HSP alignment was divided by the HSP length, and then multipliedby 100 to get the BLAST sequence identity. The HSP length typicallyincluded gaps in the alignment, but in some cases gaps were excluded.

The main Reciprocal BLAST process consists of two rounds of BLASTsearches; forward search and reverse search. In the forward search step,a reference polypeptide sequence, “polypeptide A,” from source speciesSA was BLASTed against all protein sequences from a species of interest.Top hits were determined using an E-value cutoff of 10⁻⁵ and a sequenceidentity cutoff of 35%. Among the top hits, the sequence having thelowest E-value was designated as the best hit, and considered apotential functional homolog or ortholog. Any other top hit that had asequence identity of 80% or greater to the best hit or to the originalreference polypeptide was considered a potential functional homolog orortholog as well. This process was repeated for all species of interest.

In the reverse search round, the top hits identified in the forwardsearch from all species were BLASTed against all protein sequences fromthe source species SA. A top hit from the forward search that returned apolypeptide from the aforementioned cluster as its best hit was alsoconsidered as a potential functional homolog.

Functional homologs were identified by manual inspection of potentialfunctional homolog sequences. Representative functional homologs for SEQID Nos. 2, 35, 41, 43, 44, 45, 86, 109, 135, 136, 138, 140, 141, 142,143 and to amino acids X-Y of SEQ ID NO: 140 and to amino acids X-Y ofSEQ ID NO: 140 are shown in FIGS. 1-6 and the Sequence Listing.

Example 19 Determination of Functional Homologs by Hidden Markov Models

Hidden Markov Models (HMMs) were generated by the program HMMER 2.3.2.To generate each HMM, the default HMMER 2.3.2 program parameters,configured for glocal alignments, were used.

An HMM was generated using the sequences shown in FIG. 1 as input. Thesesequences were input into the model and the HMM bit score for eachsequence is shown in the Sequence Listing. Additional sequences wereinput into the model, and the HMM bit scores for the additionalsequences are shown in the Sequence Listing. The results indicate thatthese additional sequences are functional homologs of SEQ ID NO: 86.

HMMs were also generated using the sequences shown in FIGS. 2-6 asinput. These sequences were input into the respective models and thecorresponding HMM bit score for each sequence is shown in the SequenceListing. Additional sequences were input into the models, and the HMMbit scores for the additional sequences are shown in the SequenceListing. The results indicate that these additional sequences arefunctional homologs of the groups in FIGS. 2-6.

In an alternative embodiment, the HMM is generated with the proviso thatnone of the amino acids specifically described in PCT/US2007/06544 areused. In particular the following amino acids appearing in the SequenceListing of PCT/US2007/06544 are excluded: SEQ ID NO:99, SEQ ID NO:100,SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:180, SEQ IDNO:252, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:306 andSEQ ID NO:312.

REFERENCES

The following references are cited in the Specification. Each of thereferences from the patent and periodical literature cited herein ishereby expressly incorporated in its entirety by such citation.

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The invention claimed is:
 1. A method of producing a plant havingincreased tolerance to salinity or increased tolerance to oxidativestress, said method comprising growing a plant cell transformed with anexogenous nucleic acid, said exogenous nucleic acid comprising aheterologous promoter operably linked to a nucleotide sequence encodinga polypeptide or a fragment thereof, wherein the polypeptide has 95percent or greater sequence identity to the amino acid sequence of SEQID NO: 138 and wherein said fragment has at least 93% sequence identityto the contiguous amino acids of the amino acid sequence of SEQ ID NO:140, wherein said polypeptide exhibits the functional activity of SEQ IDNO: 138, and said fragment exhibits the functional activity of SEQ IDNO: 140, and wherein overexpression of said polypeptide or said fragmentin transformed plant grown from said transformed plant cell results inan increase in the level of tolerance to salinity or oxidative stress ascompared to a control plant of the same species cultivated under thesame conditions that does not express said polypeptide or said fragment.2. The method according to claim 1, wherein the nucleotide sequenceencodes a polypeptide having 95 percent or greater sequence identity tothe amino acid sequence of SEQ ID NO:
 138. 3. The method according toclaim 1, wherein the nucleotide sequence encoding said polypeptideexhibits 95 percent or greater sequence identity to the polynucleotidesequence of SEQ ID NO:
 139. 4. The method according to claim 1, whereinthe nucleotide sequence encodes a polypeptide comprising the amino acidsequence of SEQ ID NO:
 138. 5. The method according to claim 1, whereinthe nucleotide sequence comprises the polynucleotide sequence of SEQ IDNO:139.
 6. A plant cell transformed with an exogenous nucleic acid,wherein said exogenous nucleic acid comprising a heterologous promoteroperably linked to a nucleotide sequence encoding a polypeptide or afragment thereof, wherein the polypeptide has 95 percent or greatersequence identity to the amino acid sequence of SEQ ID NO: 138 andwherein said fragment has at least 93% sequence identity to thecontiguous amino acids of the amino acid sequence of SEQ ID NO: 140,wherein said polypeptide exhibits the functional activity of SEQ ID NO:138 and said fragment exhibits the functional activity of SEQ ID NO:140, and wherein overexpression of said polypeptide or said fragment insaid transformed plant cell results in an increase in the level oftolerance to salinity or oxidative stress as compared to a control plantcell of the same species cultivated under the same conditions that doesnot express said polypeptide or said fragment.
 7. The plant cell ofclaim 6, wherein the nucleotide sequence encodes a polypeptide having 95percent or greater sequence identity to the amino acid sequence of SEQID NO:
 138. 8. The plant cell of claim 6, wherein the nucleotidesequence encoding said polypeptide exhibits 95 percent or greatersequence identity to the polynucleotide sequence of SEQ ID NO:
 139. 9. Atransgenic plant comprising the transformed plant cell of claim
 6. 10.The transgenic plant of claim 9, wherein said plant is a member of aspecies selected from the group consisting of Panicum virgatum(switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthusgiganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera(poplar), Zea mays (corn), Glycine max (soybean), Brassica napus(canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryzasativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa),Beta vulgaris (sugarbeet), and Pennisetum glaucum (pearl millet).
 11. Aseed or vegetative tissue comprising the transformed plant cell of claim6.